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Figure 5-15 The effect of single-strand DNA-binding proteins (SSB proteins) on the structure of single-stranded DNA. Because each protein molecule prefers to bind next to a previously bound molecule, long rows of this protein form on a DNA single strand. This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process. The "hairpin helices" shown in the bare, single-stranded DNA result from a chance matching of short regions of complementary nucleotide sequence.
图 5-15 单链 DNA 结合蛋白(SSB 蛋白)对单链 DNA 结构的影响。由于每个蛋白分子更倾向于结合到先前结合的分子旁边,这种蛋白在 DNA 单链上形成长行。这种协同结合使 DNA 模板变直并促进 DNA 聚合过程。裸露的单链 DNA 中显示的“发夹螺旋”是由于互补核苷酸序列的短区域偶然匹配而产生的。
releases itself from the clamp and dissociates from the template. With the help of the clamp loader, which hydrolyzes ATP as it loads a new clamp onto a primertemplate junction (Figure 5-17), this lagging-strand polymerase molecule then associates with the new clamp that is assembled on the RNA primer of the next Okazaki fragment.
释放自身并与模板解离。在夹具加载器的帮助下,夹具加载器在将新夹具加载到引物模板结合物上时水解 ATP(图 5-17),这个滞后链聚合酶分子随后与组装在下一个岡崎片段的 RNA 引物上的新夹具结合。

The Proteins at a Replication Fork Cooperate to Form a Replication Machine

Although we have discussed DNA replication as though it were performed by a set of proteins all acting independently, in reality most of these proteins are held together in a large and orderly multienzyme complex that rapidly synthesizes DNA. This complex can be likened to a tiny sewing machine composed of protein
尽管我们讨论 DNA 复制时似乎是由一组独立行动的蛋白质执行的,但实际上,这些蛋白质中的大多数被固定在一个庞大而有序的多酶复合物中,快速合成 DNA。这个复合物可以被比作由蛋白质组成的微型缝纫机。

Figure 5-16 Human single-strand binding protein bound to DNA. (A) Front view of the two DNA-binding domains of the protein (called RPA), which cover a total of eight nucleotides. Note that the DNA bases remain exposed in this protein-DNA complex. (B) Diagram showing the three-dimensional structure, with the DNA strand (orange) viewed end on. (PDB code: .)
图 5-16 人类单链结合蛋白结合到 DNA。(A) 蛋白质的两个 DNA 结合结构的正面视图(称为 RPA),总共覆盖了八个核苷酸。请注意,在这个蛋白质-DNA 复合物中,DNA 碱基保持暴露。 (B) 显示三维结构的图示,DNA 链(橙色)端面视图。(PDB 代码: .)
Figure 5-17 The sliding clamp that holds DNA polymerase on the DNA. (A) The structure of the clamp protein from E. coli, as determined by x-ray crystallography, with a DNA helix added to indicate how the protein fits around DNA (Movie 5.3). (B) Schematic illustration showing how the clamp is loaded onto DNA. The structure of the clamp loader (green) resembles a screw nut, with its threads matching the grooves of double-stranded DNA. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits, which enables it to slip around DNA. The loader then "screws" the open clamp onto double-stranded DNA until it encounters the 3 ' end of a primer, at which point the loader hydrolyzes ATP and releases the clamp, allowing it to close around the DNA. In the simplified reaction shown here, the clamp loader dissociates once the clamp has been assembled. At bacterial replication forks, the clamp loader remains bound to the polymerase so that, on the lagging strand, it is ready to assemble a new clamp at the start of each new Okazaki fragment. (A, from X.P. Kong et al., Cell 69:425-437, 1992; PDB code: 3BEP; B, adapted from B.A. Kelch et al., Science 334:1675-1680, 2011.)
图 5-17 保持 DNA 聚合酶在 DNA 上的滑动夹具。 (A) 大肠杆菌中夹具蛋白的结构,由 X 射线晶体学确定,添加了一个 DNA 螺旋以指示蛋白如何围绕 DNA (视频 5.3)。(B) 示意图显示夹具如何加载到 DNA 上。夹具加载器的结构(绿色)类似于螺母,其螺纹与双链 DNA 的凹槽相匹配。加载器结合到一个自由的夹具分子,迫使其环状亚基之间产生间隙,使其能够滑动到 DNA 周围。然后,加载器将打开的夹具“螺丝”到双链 DNA 上,直到遇到引物的 3'端,此时加载器水解 ATP 并释放夹具,使其围绕 DNA 闭合。在此处显示的简化反应中,夹具加载器在夹具组装完成后解离。在细菌复制叉处,夹具加载器保持与聚合酶结合,因此,在滞后链上,它准备在每个新的 Okazaki 片段的开始处组装一个新的夹具。(A,来源于 X.P. Kong 等人,Cell 69:425-437,1992 年; PDB 代码: 3BEP; B,改编自 B.A. Kelch 等人,Science 334:1675-1680,2011 年。)

parts and powered by nucleoside triphosphate hydrolysis. Like a sewing machine, the replication complex probably remains stationary with respect to its immediate surroundings; the DNA can be thought of as a long strip of cloth being rapidly threaded through it. Although the replication complex has been most intensively studied in E. coli and several of its viruses, a very similar complex also operates in eukaryotes, as we shall see below.
由核苷三磷酸水解提供动力的部分。就像缝纫机一样,复制复合物可能在其周围保持静止;DNA 可以被视为一条长布条,快速穿过它。尽管复制复合物在大肠杆菌及其几种病毒中得到了最深入的研究,但在真核生物中也存在一个非常相似的复合物,我们将在下文中看到。
How the different proteins at the replication fork work together in bacteria is shown in Figure 5-18. At the front of the replication fork, DNA helicase opens the DNA helix. Several identical DNA polymerase molecules work at the fork, one on the leading strand and two on the lagging strand. Whereas the DNA polymerase molecule on the leading strand can operate in a continuous fashion, the DNA polymerase molecules on the lagging-strand alternate at short intervals, using the short RNA primers made by DNA primase to begin each Okazaki fragment. The close association of all these protein components increases the efficiency of replication, and it is made possible by a folding back of the lagging strand as shown in the figure. This arrangement facilitates the loading of the polymerase clamp each time that an Okazaki fragment is synthesized: the clamp loader and the lagging-strand DNA polymerase molecule are kept in place at the replication fork even when they detach from their DNA template. The replication proteins are thus linked together into a single large unit (total molecular mass daltons), enabling DNA to be synthesized on both sides of the replication fork in a coordinated and efficient manner.
不同蛋白质在细菌复制叉处如何协同工作的示意图如图 5-18 所示。在复制叉的前端,DNA 解旋酶打开 DNA 螺旋。几个相同的 DNA 聚合酶分子在复制叉处工作,一个在前导链上,两个在滞后链上。前导链上的 DNA 聚合酶分子可以连续工作,而滞后链上的 DNA 聚合酶分子则在短间隔内交替工作,使用 DNA 引物酶制造的短 RNA 引物来开始每个 Okazaki 片段。所有这些蛋白质组分的紧密关联增加了复制的效率,这是通过滞后链的折返来实现的,如图所示。这种排列有助于在合成每个 Okazaki 片段时每次加载聚合酶夹: 即使它们从 DNA 模板上分离,夹装载器和滞后链 DNA 聚合酶分子也会保持在复制叉处。 复制蛋白质因此被连接成一个单一的大单位(总分子量 道尔顿),使得 DNA 能够在复制叉的两侧以协调和高效的方式合成。
On the lagging strand, the DNA replication machine leaves behind a series of unsealed Okazaki fragments, which still contain the RNA that primed their synthesis at their ends. As discussed earlier, this RNA is removed, and the resulting
在滞后链上,DNA 复制机器留下一系列未封闭的岡崎片段,这些片段仍然包含在其 端引导合成的 RNA。正如前面讨论的那样,这些 RNA 被去除,产生的
newly synthesized leading strand
Figure 5-18 A bacterial replication fork. (A) In this case, a single DNA polymerase molecule synthesizes the leading strand while two DNA polymerases are used - in alternating fashion-for lagging-strand DNA synthesis. All of these polymerase molecules, which are identical, are held in place at the fork by flexible "arms" that extend from the clamp loader. Additional interactions (for example, between the DNA helicase and primase) ensure that all the individual components function together as a well-coordinated protein machine (Movie 5.4). (B) An electron micrograph showing the replication machine from the bacteriophage T4 as it moves along a template synthesizing DNA behind it. (C) An interpretation of the micrograph is given in the sketch: note especially the DNA loop on the lagging strand. Apparently, during the preparation of this sample for electron microscopy, the replication proteins became partly detached from the very front of the replication fork. (B, from P.D. Chastain et al., J. Biol. Chem. 278:21276-21825, 2003. With permission from American Society for Biochemistry and Molecular Biology.)
图 5-18 细菌复制叉。 (A) 在这种情况下,单个 DNA 聚合酶分子合成领先链,而两个 DNA 聚合酶 - 交替使用 - 用于滞后链 DNA 合成。 所有这些相同的聚合酶分子都由从夹持装载器延伸的灵活“臂”固定在叉处。 额外的相互作用(例如,DNA 解旋酶和引物酶之间)确保所有单个组件作为一个协调良好的蛋白质机器一起发挥作用(电影 5.4)。 (B) 电子显微镜照片显示噬菌体 T4 的复制机器沿着模板移动,在其后合成 DNA。 (C) 在草图中给出了对显微图的解释:特别注意滞后链上的 DNA 环。 显然,在为电子显微镜制备此样品期间,复制蛋白部分从复制叉的最前端分离出来。(B,出自 P.D. Chastain 等人,J. Biol. Chem. 278:21276-21825,2003 年。获得美国生物化学与分子生物学学会许可。)
gap is filled in by DNA repair enzymes that operate behind the replication fork (see Figure 5-11).
DNA 修复酶填补了在复制叉后方运作的间隙(见图 5-11)。

DNA Replication Is Fundamentally Similar in Eukaryotes and Bacteria
DNA 复制在真核生物和细菌中基本上是相似的

Much of what we know about DNA replication was first derived from studies of purified bacterial and bacteriophage multienzyme systems capable of DNA replication in vitro. The development of these systems in the 1970s was greatly facilitated by the prior isolation of mutants in a variety of replication genes; these mutants were exploited to identify and purify the corresponding replication proteins. The first eukaryotic replication system that accurately replicated DNA in vitro was described in the mid-1980s, and mutations in genes encoding nearly all of the replication components have now been isolated and analyzed in the yeast Saccharomyces cerevisiae. As a result, much is known about the detailed enzymology of DNA replication in eukaryotes, and it is clear that the fundamental features of DNA replication-including replication-fork geometry and the use of DNA polymerases, helicases, clamps, clamp loaders, and single-strand binding proteins-are similar.
DNA 复制的许多知识最初是从对纯化的细菌和噬菌体多酶系统的研究中得出的,这些系统能够在体外进行 DNA 复制。这些系统的发展始于 1970 年代,之前已经通过多种复制基因的突变体的分离大大促进了这一过程;这些突变体被利用来识别和纯化相应的复制蛋白质。第一个能够准确在体外复制 DNA 的真核复制系统是在 1980 年代中期描述的,目前已经在酿酒酵母 Saccharomyces cerevisiae 中分离和分析了几乎所有复制组分编码基因的突变体。因此,我们对真核生物 DNA 复制的详细酶学特征有了很多了解,明显地,DNA 复制的基本特征,包括复制叉几何结构和使用的 DNA 聚合酶、解旋酶、夹具、夹具装载蛋白和单链结合蛋白等,是相似的。
Figure 5-19 Schematic diagram of a eukaryotic replication fork. Unlike the bacterial replication proteins, those from eukaryotes are thought to function largely independently, perhaps accounting for the slower speed of the eukaryotic replication fork (Movie 5.5). Note that the eukaryotic CMG helicase moves unidirectionally along the leading-strand template, whereas the bacterial helicase discussed earlier moves in one direction along the lagging-strand template (see Figure 5-18). In both cases, the DNA duplex is rapidly pried apart at the front of the moving replication fork by harnessing the energy of ATP hydrolysis.
图 5-19 真核复制叉的示意图。与细菌复制蛋白不同,真核生物的复制蛋白被认为主要独立运作,这或许解释了真核生物复制叉速度较慢的原因(见影片 5.5)。请注意,真核 CMG 解旋酶沿着前导链模板单向移动,而之前讨论的细菌解旋酶沿着滞后链模板单向移动(见图 5-18)。在两种情况下,通过利用 ATP 水解能量,DNA 双螺旋在移动复制叉前端迅速分离。
However, there are some important differences in how bacteria and eukaryotes replicate their DNA. Perhaps most important, eukaryotes use three different kinds of DNA polymerase at each replication fork (Figure 5-19). Polymerase (Pol ) synthesizes the leading strand, whereas Pol and Pol synthesize the lagging-strand Okazaki fragments. Each type of polymerase has special properties that make it well suited for its job. Pol binds to both the sliding clamp and the replicative helicase, allowing it to synthesize very long stretches of leading-strand DNA without dissociating. Pol includes DNA primase as one of its subunits, which begins all new chains by synthesizing a short length of RNA. This RNA is extended by a different subunit of Pol , which adds only about 20 nucleotides of DNA before dissociating. Finally, Pol , which is loaded in conjunction with a sliding clamp, takes over and completes synthesis of each Okazaki fragment to produce a total length of about 200 nucleotides.
然而,细菌和真核生物在复制 DNA 方面存在一些重要的差异。也许最重要的是,真核生物在每个复制叉处使用三种不同类型的 DNA 聚合酶(图 5-19)。聚合酶 (Pol )合成主链,而 Pol 和 Pol 合成滞后链的 Okazaki 片段。每种聚合酶都具有特殊的特性,使其非常适合其工作。Pol 同时结合滑动夹和复制解旋酶,使其能够在不解离的情况下合成非常长的主链 DNA 片段。Pol 将 DNA 引物酶作为其亚基之一,通过合成一小段 RNA 来开始所有新链。这段 RNA 由 Pol 的另一个亚基延伸,该亚基在解离之前仅添加约 20 个核苷酸的 DNA。最后,与滑动夹一起加载的 Pol 接管并完成每个 Okazaki 片段的合成,以产生约 200 个核苷酸的总长度。
The use of three different kinds of DNA polymerase at the replication fork is part of a trend toward higher complexity observed for eukaryotic DNA replication compared to that of bacteria. As another example, the eukaryotic single-strand binding protein is formed from three different subunits, while only a single subunit is found in bacteria. Likewise, the eukaryotic replicative helicase (known as the CMG helicase) is composed of 11 different protein subunits, while the bacterial enzyme is a hexamer of 6 identical subunits. We do not know why the eukaryotic replication machinery is so much more complex than that of bacteria; however, there are several possibilities. In eukaryotes, DNA replication must be coordinated with the elaborate process of mitosis; it must also deal with DNA packaged into nucleosomes, topics we discuss in the next part of the chapter. It is also possible that the difference in complexity between bacteria and eukaryotes largely reflects evolutionary pressure for bacteria to make do with fewer genes.
三种不同类型的 DNA 聚合酶在复制叉处的使用是观察到的真核 DNA 复制相对于细菌而言趋向更高复杂性的一部分。另一个例子是,真核单链结合蛋白由三种不同亚基组成,而细菌中只有一个亚基。同样,真核复制螺旋酶(称为 CMG 螺旋酶)由 11 种不同的蛋白亚基组成,而细菌酶是由 6 个相同亚基组成的六聚体。我们不知道为什么真核复制机制比细菌的复杂得多;然而,有几种可能性。在真核生物中,DNA 复制必须与有丝分裂的复杂过程协调进行;它还必须处理包装成核小体的 DNA,这是我们在本章的下一部分讨论的主题。细菌和真核生物之间复杂性差异的原因可能主要反映了细菌需要利用更少基因的进化压力。
Another important distinction between eukaryotic and bacterial replication protein complexes lies in the detailed structures of their individual protein

components. With the exception of the sliding clamp, the replication proteins in bacteria have completely different structures and amino acid sequences than those of their eukaryotic counterparts. The simplest interpretation of this surprising fact is that, over hundreds of millions of years, the DNA replication machinery in eukaryotes and bacteria evolved independently, yet converged on the same basic mechanisms. This situation is in contrast to other fundamental processes in the cell, such as transcription and translation, where the fundamental components (RNA polymerase and the ribosome) are very similar between bacteria and eukaryotes-and where the structures are conserved from an ancient, common ancestor.
除了滑动夹具外,细菌中的复制蛋白与真核生物的对应物在结构和氨基酸序列上完全不同。对这一令人惊讶的事实最简单的解释是,在数亿年的时间里,真核生物和细菌中的 DNA 复制机制独立演化,但最终采用了相同的基本机制。这种情况与细胞中的其他基本过程形成对比,比如转录和翻译,在这些过程中,基本组分(RNA 聚合酶和核糖体)在细菌和真核生物之间非常相似,结构保留自古老的共同祖先。

A Strand-directed Mismatch Repair System Removes Replication Errors That Remain in the Wake of the Replication Machine

Because bacteria such as E. coli are capable of dividing once every 30 minutes, it is relatively easy to screen large populations to find a rare mutant cell that is altered in a specific process. One interesting class of mutants consists of those with alterations in so-called mutator genes, which greatly increase the rate of spontaneous mutation. Not surprisingly, one such mutant makes a defective form of the -to-5' proofreading exonuclease that is a part of the DNA polymerase enzyme (see Figures 5-8 and 5-9). The mutant DNA polymerase no longer proofreads effectively, and many replication errors that would otherwise have been removed accumulate in the DNA.
由于细菌如大肠杆菌能够每 30 分钟分裂一次,因此相对容易筛选大量群体以找到在特定过程中发生改变的罕见突变细胞。一个有趣的突变体类别包括那些在所谓的突变基因中发生改变的细胞,这些基因大大增加了自发突变的速率。毫不奇怪,这样的一个突变体制造了 DNA 聚合酶酶中的一种缺陷形式的 -to-5'校对外切酶(见图 5-8 和 5-9)。突变的 DNA 聚合酶不再有效地进行校对,许多本应被移除的复制错误在 DNA 中积累。
The study of other . coli mutants exhibiting abnormally high mutation rates uncovered an additional proofreading system, common to all cells on Earth, that removes those rare replication errors that were made by the polymerase and missed by its proofreading exonuclease. These errors leave mismatched base pairs behind the replication fork, which are subsequently recognized and corrected by a strand-directed mismatch repair system. This system picks out mismatches from normal DNA by monitoring their potential to distort the DNA double helix, which is greatly increased by the misfit between noncomplementary base pairs. However, if the repair system simply recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched nucleotides, it would mistakenly "correct" the original template strand to match the error exactly half the time, thereby failing to lower the overall error rate. To be effective, such a proofreading system must be able to remove only the nucleotide on the newly synthesized strand, where the error occurred.
对其他大肠杆菌突变体的研究揭示了一种额外的校对系统,这种系统普遍存在于地球上的所有细胞中,它能够去除由聚合酶产生但被其校对外切酶忽略的那些罕见的复制错误。这些错误会在复制叉点之后留下不匹配的碱基对,随后会被一个链向错配修复系统识别并纠正。该系统通过监测错配碱基对扭曲 DNA 双螺旋的潜力来从正常 DNA 中挑选出错配。由于非互补碱基对之间的不匹配会大大增加 DNA 双螺旋的扭曲程度。然而,如果修复系统仅仅识别新复制的 DNA 中的错配并随机纠正两个错配核苷酸中的一个,那么它将错误地“纠正”原始模板链以使其与错误完全匹配的概率达到一半,从而无法降低整体错误率。为了有效,这样的校对系统必须能够仅去除新合成链上发生错误的核苷酸。
The strand-distinction mechanism used by the mismatch proofreading system in E. coli depends on the methylation of selected A residues in the DNA. Methyl groups are added to all A residues in the sequence GATC, but not until some time after the GATC has been synthesized. As a result, the only unmethylated GATC sequences lie in the newly synthesized strands just behind a replication fork. The recognition of these unmethylated GATCs (which are base-paired to methylated GATCs) allows the new DNA strands to be transiently distinguished from old ones, as required if their mismatches are to be selectively removed. The five-step error-correction process involves recognition of a mismatch, identification of the newly synthesized strand, excision of the portion containing the misincorporated nucleotide, resynthesis of the excised segment using the old strand as a template, and ligation to seal the DNA backbone. This strand-directed mismatch repair system reduces the number of errors made during DNA replication by an additional factor of 100-1000 (see Table 5-1, p. 260).
大肠杆菌中的错配校对系统使用的链区别机制取决于 DNA 中选择性 A 残基的甲基化。甲基基团被添加到序列 GATC 中的所有 A 残基,但直到 GATC 合成后的一段时间才添加。因此,唯一未甲基化的 GATC 序列位于复制叉后方刚合成的链中。识别这些未甲基化的 GATC(与甲基化的 GATC 成对)使得新的 DNA 链能够暂时与旧的区分开来,这是必要的,以便选择性地去除它们的错配。这个五步错误校正过程包括识别错配、识别新合成链、切除含有错误插入核苷酸的部分、使用旧链作为模板重新合成被切除的片段,以及连接以封闭 DNA 骨架。这个链定向的错配修复系统通过额外减少 100-1000 倍 DNA 复制过程中的错误数量(见表 5-1,第 260 页)。
A similar mismatch proofreading system functions in eukaryotic cells, but it uses a different way to distinguish the newly synthesized DNA strands from the parent strands. On the lagging strand, the newly synthesized DNA will contain transient single-strand gaps before the series of Okazaki fragments are processed and ligated together. Each gap will usually carry a sliding clamp, which remains on the DNA after the DNA polymerase has dissociated from it to begin the next fragment. Together, the clamp and the single-strand break signal to the mismatch
在真核细胞中,存在类似的不匹配校对系统,但它使用不同的方式来区分新合成的 DNA 链与母链。在滞后链上,新合成的 DNA 将在经过一系列 Okazaki 片段的处理和连接之前包含短暂的单链间隙。每个间隙通常会携带一个滑动夹具,该夹具在 DNA 聚合酶从 DNA 上解离以开始下一个片段后仍保留在 DNA 上。夹具和单链断裂一起向不匹配信号。
Figure 5-20 Strand-directed mismatch repair in eukaryotes. (A) The MutS protein binds to a mismatched base pair, recruits the MutL protein, and the complex scans the nearby DNA for a gap and a sliding clamp whose orientation determines which strand is to be cut and its nucleotides replaced. When these are encountered, MutL is activated and begins to cleave the DNA. In most organisms, MutL is joined by another nuclease and, together, they remove the newly synthesized DNA starting at the gap and extending past the mismatch. The gap is then filled in by DNA polymerase and sealed by DNA ligase. (B) The structure of the MutS protein bound to a DNA mismatch. This protein is a dimer, which grips the DNA double helix as shown, kinking the DNA at the mismatched base pair. It seems that the MutS protein scans the DNA for mismatches by testing for sites that can be readily kinked, which are those with an abnormal base pair. (PDB code: 1EWQ.)
图 5-20 有向链错配修复在真核生物中的作用。(A) MutS 蛋白结合到错配碱基对,招募 MutL 蛋白,复合物扫描附近的 DNA 以寻找缺口和一个滑动夹具,其方向决定应切割哪条链以及其核苷酸被替换。当遇到这些情况时,MutL 被激活并开始切割 DNA。在大多数生物中,MutL 会与另一种核酸酶结合,一起去除从缺口开始并延伸过错配的新合成 DNA。然后,DNA 聚合酶填补缺口并由 DNA 连接酶封闭。(B) MutS 蛋白与 DNA 错配结合的结构。该蛋白是二聚体,如图所示紧握 DNA 双螺旋,在错配碱基对处使 DNA 弯曲。MutS 蛋白似乎通过测试可轻松弯曲的位点来扫描 DNA 中的错配,这些位点是具有异常碱基对的位点。(PDB 代码:1EWQ)。
repair proteins to correct the mismatch using the parent DNA strand as the template (Figure 5-20).
使用母本 DNA 链作为模板,修复蛋白质来纠正错配(图 5-20)。
The two faces of the clamp differ, and the clamp loader always loads the clamp in the same orientation with respect to the end of the previously synthesized Okazaki fragment. Because all the clamps on the DNA "face" in the same direction relative to the replication process, the oriented clamps can be used by the mismatch repair machinery to distinguish newly synthesized DNA from parent DNA. It is not known for certain how strand discrimination occurs on the leading strand (where gaps in newly synthesized DNA should be rare), but because oriented sliding clamps are also left behind by the leading-strand polymerase, they can signal old from new DNA in the same way that they do on the lagging strand. The recent discovery of a correction system that removes misincorporated ribonucleotides suggests a further possibility for distinguishing newly synthesized DNA from parent DNA, as we discuss in the next section.
夹具的两个面不同,夹具加载器总是以与先前合成的 Okazaki 片段的 端相同的方向加载夹具。由于 DNA 上的所有夹具在相对于复制过程的同一方向上,“面向”,定向的夹具可以被错配修复机制用来区分新合成的 DNA 和母本 DNA。目前尚不清楚如何在前导链上进行链鉴别(在新合成的 DNA 中缺陷应该很少),但由于定向的滑动夹具也被前导链聚合酶留下,它们可以以与在滞后链上相同的方式来区分新旧 DNA。最近发现了一个纠正系统,可以去除错误插入的核糖核苷酸,这提出了一个进一步的可能性,用于区分新合成的 DNA 和母本 DNA,我们将在下一节中讨论。
Mismatch correction is crucial for all cells; its importance for humans is seen in individuals who inherit one defective copy of a mismatch repair gene (along

with a functional gene on the other copy of the chromosome). These individuals have a marked predisposition for certain types of cancers. For example, in a type of colon cancer called hereditary nonpolyposis colorectal cancer (HNPCC), a spontaneous deleterious mutation of the one functional gene will produce a clone of somatic cells that, because they are deficient in mismatch proofreading, accumulate mutations unusually rapidly. Because most cancers arise in cells that have accumulated many mutations (as discussed in Chapter 20), cells deficient in mismatch proofreading have a greatly enhanced chance of becoming cancerous. Fortunately, most of us inherit two good copies of each gene that encodes a mismatch proofreading protein; this protects us, because it is highly unlikely that both copies will become mutated in the same cell.
拥有染色体另一拷贝上的一个功能基因)。这些个体对某些类型的癌症有明显的易感性。例如,在一种称为遗传性非息肉性结直肠癌(HNPCC)的结肠癌类型中,一个功能基因的自发有害突变将产生一群体细胞克隆,因为它们缺乏错配修复,导致异常快速地积累突变。因为大多数癌症发生在已经积累了许多突变的细胞中(如第 20 章所讨论的),缺乏错配修复的细胞极有可能发展为癌症。幸运的是,我们大多数人继承了每个编码错配修复蛋白的基因的两个良好拷贝;这保护了我们,因为两个拷贝在同一细胞中发生突变的可能性极低。

The Accidental Incorporation of Ribonucleotides During DNA Replication Is Corrected
DNA 复制过程中意外包含核糖核苷酸的错误被纠正

We have seen that cells have several ways to correct mistakes where the wrong deoxynucleotide has been incorporated in newly replicated DNA. Occasionally, however, DNA polymerases make a different kind of mistake, one that is not caused by improper base-pairing: in this case, they accidently incorporate a ribonucleotide instead of a deoxyribonucleotide. These molecules differ by a single group in the sugar portion of the nucleotide. Yet, when incorporated into DNA, they weaken the DNA chain at that point, rendering it highly susceptible to breakage. If left unrepaired, these "weak links" would cause high mutation rates and genome rearrangements. Even if it does not cause a break, an incorporated ribonucleotide distorts the DNA double helix and can stall some polymerases during the next cycle of DNA replication.
我们已经看到细胞有几种纠正错误的方式,其中错误的脱氧核苷酸已经被合并到新复制的 DNA 中。然而,偶尔,DNA 聚合酶会犯一种不同类型的错误,这种错误不是由于不正确的碱基配对引起的:在这种情况下,它们意外地将一个核糖核苷酸而不是脱氧核苷酸合并进去。这些分子在核苷酸的糖部分只有一个 基团的差异。然而,当它们被合并到 DNA 中时,它们会在那一点削弱 DNA 链,使其极易断裂。如果不加修复,这些“薄弱环节”将导致高突变率和基因组重排。即使它不引起断裂,合并的核糖核苷酸也会扭曲 DNA 双螺旋,并且在下一个 DNA 复制周期中会使一些聚合酶停滞。
Although DNA polymerases much prefer deoxyribonucleotides over ribonucleotides (by a factor of about a million), the concentration of ribonucleotides in the cell is much higher than that of their deoxy counterparts, as much as 500 -fold for ATP, which has many different uses in the cell. This concentration imbalance means that a ribonucleotide is accidentally incorporated approximately once per several thousand nucleotides of DNA synthesized. These mistakes are corrected by specific nucleases that cleave the DNA chain when they encounter a ribonucleotide, leading to the excision of the ribonucleotide and its replacement by DNA, much in the same way that RNA primers are replaced by DNA to complete lagging-strand synthesis (see Figure 5-11). Because this repair process produces gaps only in newly synthesized DNA, it has been proposed that these transient lesions help the mismatch repair system "know" which strand to repair; in particular, these cues may be especially important on the leading strand.
尽管 DNA 聚合酶更偏爱脱氧核苷酸而非核糖核苷酸(大约高出一百万倍),但细胞中核糖核苷酸的浓度远高于其脱氧核苷酸对应物,例如 ATP 的浓度高达 500 倍,而 ATP 在细胞中有许多不同的用途。这种浓度不平衡意味着在合成 DNA 时,大约每几千个核苷酸中就会意外地插入一个核糖核苷酸。这些错误会被特定的核酸酶纠正,当它们遇到核糖核苷酸时会切断 DNA 链,导致核糖核苷酸的切除并被 DNA 替换,就像 RNA 引物被 DNA 替换以完成滞后链合成一样(见图 5-11)。由于这种修复过程只在新合成的 DNA 中产生间隙,因此有人提出这些短暂的损伤有助于错配修复系统“知道”应该修复哪条链;特别是在前导链上,这些提示可能尤为重要。

DNA Topoisomerases Prevent DNA Tangling
DNA 拓扑异构酶防止 DNA 缠结

During Replication 在复制过程中

As a replication fork moves along double-stranded DNA, it creates what has been called the "winding problem." The two parent strands that are wound around each other must be unwound and separated for replication to occur. For every 10 nucleotide pairs replicated at the fork, one complete turn of the parent double helix must be unwound. In principle, this unwinding can be achieved by rapidly rotating the entire chromosome ahead of a moving fork; however, this is energetically highly unfavorable (particularly for long chromosomes). Instead, the DNA in front of a replication fork becomes overwound (Figure 5-21). This overwinding is continually relieved by enzymes known as DNA topoisomerases.
当复制叉沿着双链 DNA 移动时,它产生了所谓的“缠绕问题”。缠绕在一起的两个亲本链必须被解开和分离才能进行复制。在复制叉处复制的每 10 个核苷酸对,必须解开一个完整的亲本双螺旋的转数。原则上,这种解旋可以通过快速旋转整个染色体以超前于移动的叉来实现;然而,这在能量上是非常不利的(特别是对于长染色体)。相反,复制叉前面的 DNA 变得过度缠绕(图 5-21)。这种过度缠绕不断地被称为 DNA 拓扑异构酶的酶缓解。
A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.
DNA 拓扑异构酶可以被视为可逆核酸酶,它在 DNA 骨架磷酸酯上以共价方式加入自身,从而在 DNA 链中断一个磷酸二酯键。这个反应是可逆的,当蛋白质离开时,磷酸二酯键重新形成。
One type of topoisomerase, called topoisomerase I, produces a transient single-strand break; this break in the phosphodiester backbone allows the
一种拓扑异构酶,称为拓扑异构酶 I,产生一种瞬时的单链断裂;磷酸二酯骨架中的这种断裂允许
(C) torsional stress ahead of the helicase is relieved by free rotation of DNA around the phosphodiester bond opposite the single-strand break; the same DNA topoisomerase molecule that produced the break reseals it
(C) 螺旋酶前方的扭转应力通过 DNA 围绕单链断裂对面的磷酸二酯键的自由旋转得以缓解;产生断裂的同一 DNA 拓扑异构酶分子将其重新封闭
two sections of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point (Figure 5-22). Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. As a result, DNA replication can occur with the rotation of only a short length of helix-the part just ahead of the fork. Because the covalent linkage that joins the DNA topoisomerase protein to a DNA phosphate retains the energy of the cleaved phosphodiester bond, resealing is rapid and does not require additional energy input. In this respect, the rejoining mechanism differs from that catalyzed by the enzyme DNA ligase, discussed previously (see Figure 5-12).
DNA 螺旋的两个部分在切口两侧可以自由旋转,相对于彼此,使用切口对面链中的磷酸二酯键作为旋转点(图 5-22)。 DNA 螺旋中的任何张力都会驱动这种旋转,以减轻张力的方向旋转。因此,DNA 复制可以仅通过螺旋的短长度旋转进行 - 就在叉前方的部分。由于连接 DNA 拓扑异构酶蛋白质与 DNA 磷酸的共价键保留了切割的磷酸二酯键的能量,重新封闭是快速的,不需要额外的能量输入。在这方面,重新连接机制与之前讨论的酶 DNA 连接酶催化的机制不同(参见图 5-12)。
A second type of DNA topoisomerase, topoisomerase II, forms a covalent linkage to both strands of the DNA helix at the same time, making a transient double-strand break in the helix. These enzymes are activated by sites on chromosomes where two double helices cross over each other, such as those generated by supercoiling in front of a replication fork (see Figure 5-21B). As illustrated in Figure 5-23, once a topoisomerase II molecule binds to such a crossing site, the protein uses ATP hydrolysis to perform the following set of reactions: (1) it breaks one double helix reversibly to create a DNA "gate"; (2) it causes the second, nearby double helix to pass through this opening; and (3) it then reseals the break and dissociates from the DNA. At crossover points generated by supercoiling, passage of the double helix through the gate occurs in the direction that will reduce supercoiling. In this way, type II topoisomerases-like type I topoisomerases-can relieve the overwinding tension generated in front of a replication fork.
DNA 拓扑异构酶的第二种类型,拓扑异构酶 II,同时与 DNA 螺旋的两条链形成共价连接,使螺旋中出现瞬时的双链断裂。这些酶被激活于染色体上的交叉点,例如由复制叉前的超螺旋所产生的那些交叉点(见图 5-21B)。正如图 5-23 所示,一旦拓扑异构酶 II 分子结合到这样一个交叉点,蛋白质利用 ATP 水解来执行以下一系列反应:(1)它可可逆地断裂一个双螺旋以创建 DNA“门”;(2)它导致第二个附近的双螺旋通过这个开口;(3)然后重新封闭断裂并与 DNA 解离。在由超螺旋产生的交叉点上,双螺旋通过门的通过方向是为了减少超螺旋。通过这种方式,II 型拓扑异构酶-就像 I 型拓扑异构酶一样-可以缓解复制叉前产生的过度绞紧张力。
Their reaction mechanism also allows type II DNA topoisomerases to efficiently separate any intertwined DNA molecules. This ability of topoisomerase II is especially important for preventing the severe DNA tangling problems that would otherwise arise from DNA replication. This role is nicely illustrated by mutant yeast cells that produce, in place of the normal topoisomerase II, a version that is inactive above . When the mutant cells are warmed to this temperature, their daughter chromosomes remain intertwined after DNA replication and are unable to separate. The enormous usefulness of topoisomerase II for untangling
它们的反应机制还允许 II 型 DNA 拓扑异构酶高效地分离任何缠绕在一起的 DNA 分子。拓扑异构酶 II 的这种能力对于防止由 DNA 复制引起的严重 DNA 缠结问题尤为重要。这一作用由突变酵母细胞很好地说明,这些细胞产生了一个在 以上无法活动的版本,代替了正常的拓扑异构酶 II。当这些突变细胞被加热到这个温度时,它们的子染色体在 DNA 复制后仍然缠绕在一起,无法分离。拓扑异构酶 II 在解开缠结问题方面的巨大用处得到了很好的展示。

Figure 5-21 The "winding problem" that arises during DNA replication. (A) For a bacterial replication fork moving at 500 nucleotides per second, the parent DNA helix ahead of the fork must rotate at about 50 revolutions per second. The brackets represent about 20 turns of DNA. (B) If the ends of the DNA double helix remain fixed (or difficult to rotate), tension builds up in front of the replication fork as it becomes overwound. Some of this tension can be taken up by supercoiling, whereby the DNA double helix twists around itself. However, if the tension continues to build up, the replication fork will eventually stop because further unwinding requires more energy than the DNA helicase at the fork can provide. (C) DNA topoisomerases relieve this stress by generating temporary singlestrand breaks in the DNA, which allow rapid rotation around the single strands opposite the break.
图 5-21 DNA 复制过程中出现的“缠绕问题”。(A) 对于以每秒 500 个核苷酸移动的细菌复制叉,叉前的母体 DNA 螺旋必须以大约每秒 50 次的速度旋转。括号代表大约 20 个 DNA 转弯。(B) 如果 DNA 双螺旋的末端保持固定(或难以旋转),则在复制叉前方会积聚张力,因为它变得过度缠绕。其中一些张力可以通过超螺旋来消除,即 DNA 双螺旋围绕自身扭曲。然而,如果张力继续积聚,复制叉最终将停止,因为进一步展开需要比叉处 DNA 解旋酶提供的能量更多。(C) DNA 拓扑异构酶通过在 DNA 中产生临时单链断裂来缓解这种压力,这允许在断裂相对的单链周围快速旋转。

the original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible

spontaneous re-formation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase
磷酸二酯键的自发重组再生了 DNA 螺旋和 DNA 拓扑异构酶
Figure 5-22 The reversible DNA nicking reaction catalyzed by a DNA topoisomerase I enzyme. As indicated, these enzymes transiently form a single covalent bond with DNA; this allows free rotation of the DNA around the covalent backbone bonds linked to the blue phosphate. On reversal of the reaction, the enzyme and the DNA are restored, the only difference being the relaxation of tension in the DNA.
图 5-22 DNA 拓扑异构酶 I 酶催化的可逆 DNA 切割反应。如图所示,这些酶会暂时与 DNA 形成单一共价键;这使得 DNA 围绕与蓝色磷酸盐相连的共价骨架键自由旋转。在反应逆转时,酶和 DNA 得以恢复,唯一的区别是 DNA 中张力的放松。

the topoisomerase gate opens to let the

second DNA helix pass
第二个 DNA 螺旋经过

reversal of the covalent attachment of the topoisomerase restores an intact orange double helix
two DNA double helices that are separated
两个被分开的 DNA 双螺旋
opoisomerase ecognizes the entanglement and makes a reversible covalent attachment o the two opposite strands of one of the double helices (orange) ing a double trand break and forming a protein gate

chromosomes before mitosis begins can readily be appreciated by anyone who has struggled to remove a severe tangle from a fishing line-or from a large ball of thread-without the aid of scissors.

Summary 摘要

DNA replication takes place at a -shaped structure called a replication fork. Self-correcting DNA polymerase enzymes catalyze nucleotide polymerization in a 5'-to-3' direction, copying a DNA template strand with remarkable fidelity. Because the two strands of a DNA double helix are antiparallel, this 5'-to-3' DNA synthesis can take place continuously on only one of the strands at a replication fork (the leading strand). On the lagging strand, short DNA fragments must be made by a "backstitching" process. Because the self-correcting DNA polymerases cannot start a new chain, these lagging-strand DNA fragments are primed by short RNA primer molecules that are subsequently erased and replaced with DNA.
DNA 复制发生在一个称为复制叉的 形结构上。自我校正的 DNA 聚合酶酶催化核苷酸在 5'-到 3'方向上的聚合,以非凡的准确性复制 DNA 模板链。由于 DNA 双螺旋的两条链是反平行的,这种 5'-到 3'的 DNA 合成只能在复制叉上的一条链上连续进行(领先链)。在滞后链上,必须通过“回缝”过程制作短的 DNA 片段。由于自我校正的 DNA 聚合酶无法启动新链,这些滞后链 DNA 片段由短的 RNA 引物分子引导,随后被擦除并替换为 DNA。
DNA replication requires the cooperation of many proteins. These include (1) DNA polymerases and DNA primases to catalyze nucleoside triphosphate polymerization; (2) DNA helicases and single-strand DNA-binding (SSB) proteins to help in opening up the DNA helix so that it can be copied; (3) clamps and clamp loaders to enable DNA polymerases to copy longer stretches of DNA; (4) DNA ligases and enzymes that degrade RNA primers to seal together the discontinuously synthesized lagging-strand DNA fragments; and (5) DNA topoisomerases to help to relieve helical winding and DNA tangling problems. Many of these proteins associate with each other at a replication fork to form a highly efficient "replication machine," through which the activities and spatial movements of the individual components are coordinated.
DNA 复制需要许多蛋白质的合作。这些蛋白质包括(1)DNA 聚合酶和 DNA 引物酶,用于催化核苷酸三磷酸聚合;(2)DNA 解旋酶和单链 DNA 结合蛋白(SSB 蛋白),帮助打开 DNA 螺旋以便进行复制;(3)夹具和夹具加载器,使 DNA 聚合酶能够复制更长的 DNA 片段;(4)DNA 连接酶和降解 RNA 引物的酶,用于封闭不连续合成的滞后链 DNA 片段;以及(5)DNA 拓扑异构酶,帮助缓解螺旋缠绕和 DNA 缠结问题。这些蛋白质中的许多在复制叉处相互结合,形成一个高效的“复制机器”,通过这个机器,各个组分的活动和空间移动得到协调。
The self-correcting DNA polymerases make mistakes only rarely when copying DNA; when they do, a variety of enzymes inspect the DNA shortly after it is made and correct any mishaps. Given the number of proteins dedicated to the task, copying DNA with extreme accuracy is clearly of great importance to all cells on Earth.
自我校正的 DNA 聚合酶在复制 DNA 时很少犯错;当它们犯错时,各种酶会在 DNA 制成后不久检查 DNA 并纠正任何错误。鉴于专门从事这项任务的蛋白质数量,以极高的准确性复制 DNA 显然对地球上所有细胞都至关重要。

染色体中 DNA 复制的启动和完成

We have seen how a set of replication proteins rapidly and accurately generates two daughter DNA double helices behind a replication fork. But how is this replication machinery assembled in the first place, and how are replication forks created on an intact, double-strand DNA molecule? In this part of the chapter, we discuss how cells initiate DNA replication and how they carefully regulate this process to ensure that it takes place only at the proper time and chromosomal sites. We also discuss special problems that the replication machinery in eukaryotic cells must overcome including the need to replicate the enormously long DNA molecules found in eukaryotic chromosomes, as well as the need to copy DNA molecules that are tightly complexed with nucleosomes.
我们已经看到,一组复制蛋白迅速而准确地在复制叉后面生成两个 DNA 双螺旋的女儿。但是,这个复制机器是如何首次组装的,以及如何在完整的双链 DNA 分子上创建复制叉呢?在本章的这一部分中,我们讨论细胞如何启动 DNA 复制以及它们如何仔细调控这个过程,以确保它只在适当的时间和染色体位点发生。我们还讨论真核细胞中复制机器必须克服的特殊问题,包括需要复制真核染色体中发现的巨大长 DNA 分子,以及需要复制与核小体紧密结合的 DNA 分子。

DNA Synthesis Begins at Replication Origins
DNA 合成始于复制起源

As discussed previously, the DNA double helix is normally very stable: the two DNA strands are locked together firmly by the hydrogen bonds formed between the bases on each strand. To begin DNA replication, the double helix must first be opened up and the two strands separated to expose unpaired bases. As we shall see, the process of DNA replication is begun by special initiator proteins that bind to double-stranded DNA and pry the two strands apart, breaking the hydrogen bonds between the bases.
正如前面讨论的那样,DNA 双螺旋结构通常非常稳定:两条 DNA 链通过各自链上碱基之间形成的氢键牢固地锁在一起。要开始 DNA 复制,首先必须打开双螺旋结构,将两条链分开以暴露未配对的碱基。正如我们将看到的,DNA 复制的过程是由特殊的启动蛋白质开始的,它们结合到双链 DNA 上并将两条链分开,打破碱基之间的氢键。
The positions at which the DNA helix is first opened are called replication origins (Figure 5-24). In simple cells like those of bacteria or budding yeast, origins are specified by DNA sequences several hundred nucleotide pairs in
DNA 螺旋首次打开的位置称为复制起点(图 5-24)。在细菌或酵母等简单细胞中,起点由数百个核苷酸对的 DNA 序列指定。
Figure 5-24 A replication bubble formed by replication-fork initiation. This diagram outlines the major steps in the initiation of replication forks at replication origins. In the last step, two replication forks move away from each other, separated by an expanding replication bubble.
图 5-24 由复制叉起始形成的复制泡泡。该图解释了在复制起源处复制叉启动的主要步骤。在最后一步中,两个复制叉相互远离,被一个不断扩张的复制泡泡分隔。

length. This DNA contains both short sequences that attract initiator proteins and stretches of DNA that are especially easy to open. We saw in Figure 4-5A that an A-T base pair is held together by fewer hydrogen bonds than is a G-C base pair. Therefore, DNA rich in A-T base pairs is relatively easy to pull apart, and regions of DNA enriched in A-T base pairs are typically found at replication origins.
长度。这段 DNA 包含吸引启动蛋白的短序列和特别容易打开的 DNA 片段。如图 4-5A 所示,A-T 碱基对由比 G-C 碱基对更少的氢键保持在一起。因此,富含 A-T 碱基对的 DNA 相对容易分开,并且富含 A-T 碱基对的 DNA 区域通常在复制起源处找到。
Although the basic process of replication-fork initiation depicted in Figure 5-24 is fundamentally the same for bacteria and eukaryotes, the detailed way in which this process is performed and regulated differs considerably between these two groups of organisms. We first consider the case in bacteria and then turn to the more complex situation found in yeasts, mammals, and other eukaryotes.
尽管图 5-24 中描绘的复制叉起始的基本过程在细菌和真核生物中基本相同,但在这两组生物中,这一过程的执行和调控方式有很大差异。我们首先考虑细菌中的情况,然后转向酵母、哺乳动物和其他真核生物中发现的更为复杂的情况。

Bacterial Chromosomes Typically Have a Single Origin of DNA Replication
细菌染色体通常只有一个 DNA 复制起点

The genome of . coli is contained in a single circular DNA molecule of nucleotide pairs. DNA replication begins at a single origin of replication, and the two replication forks assembled there proceed (at approximately 1000 nucleotides per second) in opposite directions until they meet up roughly halfway around the chromosome (Figure 5-25). The only point at which E. coli can control DNA replication is initiation: once the forks have been assembled at the origin, they synthesize DNA at a relatively constant speed until replication is finished. Therefore, it is not surprising that the initiation step of DNA replication is tightly regulated. The process begins when specialized initiator proteins (in their ATP-bound state) bind in multiple copies to specific DNA sites located at the replication origin, wrapping the DNA around the proteins to form a large protein-DNA filament that introduces torsional stress on the DNA double helix (Figure 5-26). This stress is partially relieved by melting of the adjacent AT-rich sequences. The protein-DNA complex then attracts two DNA helicases, each bound to a helicase loader, and these are placed-facing in opposite directionsaround adjacent DNA single strands whose bases have been exposed by the assembly of the initiator protein-DNA complex. The helicase loader is analogous to the clamp loader we encountered earlier; it has the additional job of keeping the helicase in an inactive form until it is properly loaded. Once the helicases are properly positioned on DNA, the loaders dissociate and the helicases begin to unwind DNA, exposing enough single-stranded DNA for DNA primases to synthesize the first RNA primers. This quickly leads to the assembly of the remaining replication proteins to create two replication forks that move in opposite directions away from the replication origin, each synthesizing new DNA as they travel.
大肠杆菌的基因组包含在一个单环 DNA 分子中,共有 个核苷酸对。DNA 复制始于单个复制起始点,两个在那里组装的复制叉以相反方向进行(大约每秒 1000 个核苷酸),直到它们大约在染色体的中间位置相遇(图 5-25)。大肠杆菌唯一能够控制 DNA 复制的时机是起始阶段:一旦复制叉在起始点组装完成,它们以相对恒定的速度合成 DNA,直到复制完成。因此,DNA 复制的起始阶段受到严格调控并不令人意外。该过程始于专门的启动蛋白(处于其 ATP 结合状态)以多份结合到特定 DNA 位点位于复制起始点周围,将 DNA 缠绕在蛋白质周围形成大型蛋白质-DNA 螺旋,对 DNA 双螺旋施加扭转应力(图 5-26)。相邻富含 AT 序列的熔解部分部分缓解了这种应力。 蛋白质-DNA 复合物随后吸引两个 DNA 解旋酶,每个解旋酶与一个解旋酶加载器结合,它们被放置在相对方向上,围绕着由启动蛋白质-DNA 复合物组装而暴露出碱基的相邻 DNA 单链。解旋酶加载器类似于我们之前遇到的夹具加载器;它的额外工作是保持解旋酶处于非活性形式,直到正确加载为止。一旦解旋酶正确定位在 DNA 上,加载器解离,解旋酶开始解开 DNA,暴露出足够的单链 DNA,以便 DNA 引物酶合成第一个 RNA 引物。这迅速导致其余复制蛋白质的组装,形成两个复制叉,沿着远离复制起点的相反方向移动,每个在移动时合成新的 DNA。
In E. coli, the interaction of the initiator proteins with the replication origin is carefully regulated, with initiation occurring only when sufficient nutrients are available for the bacterium to complete an entire round of replication. Initiation is also controlled to ensure that only one round of DNA replication occurs for each cell division. After replication is initiated, the initiator protein is inactivated by hydrolysis of its bound ATP molecule, and the origin of replication experiences a refractory period. The refractory period is caused by a delay in the methylation of newly incorporated A nucleotides in the origin (Figure 5-27). Initiation cannot occur again until the A's are methylated and the initiator protein is restored to its ATP-bound state, conditions that are met only when the cell is capable of carrying out a new round of DNA replication.
在大肠杆菌中,启动蛋白与复制起源的相互作用受到精心调控,只有在细菌有足够的营养来完成整个复制周期时才会发生启动。启动也受到控制,以确保每次细胞分裂只发生一轮 DNA 复制。在启动复制后,通过水解其结合的 ATP 分子来使启动蛋白失活,并且复制起源经历一段不敏感期。这段不敏感期是由于起源中新合并的 A 核苷酸甲基化延迟引起的(图 5-27)。只有当 A 核苷酸被甲基化并且启动蛋白恢复到其结合 ATP 的状态时,启动才能再次发生,这些条件只有在细胞能够进行新一轮 DNA 复制时才能满足。

Eukaryotic Chromosomes Contain Multiple Origins of Replication

We have seen how two replication forks begin at a single replication origin in bacteria and proceed in opposite directions, moving away from the origin until all of the DNA in the single circular chromosome is replicated. The bacterial genome is sufficiently small for these two replication forks to duplicate the genome in about
我们已经看到,在细菌中,两个复制叉从单个复制起源开始,并朝相反方向移动,远离起源,直到单个圆形染色体中的所有 DNA 被复制。细菌基因组足够小,使得这两个复制叉能够在大约
Figure 5-25 DNA replication of a bacterial genome. It takes E. coli about 30 minutes to duplicate its genome of nucleotide pairs. For simplicity, Okazaki fragments are not shown on the lagging strand.
图 5-25 细菌基因组的 DNA 复制。大肠杆菌大约需要 30 分钟来复制其由 个核苷酸对组成的基因组。为简单起见,滞后链上未显示岡崎片段。
30 minutes. Because of the much greater size of most eukaryotic chromosomes, a different strategy is required to allow their replication in a timely manner.
30 分钟。由于大多数真核染色体的尺寸更大,需要采用不同的策略来确保它们能够及时复制。
A method for determining the general pattern of eukaryotic chromosome replication was developed in the early 1960s that is similar to the strategy we saw earlier for visualizing bacterial replication (see Figure 5-6). Human cells growing
20 世纪 60 年代初期开发了一种确定真核染色体复制一般模式的方法,类似于我们早前看到的用于可视化细菌复制的策略(见图 5-6)。正在生长的人类细胞
Figure 5-26 The proteins that initiate DNA replication in bacteria. The mechanism shown was established by studies in vitro with mixtures of highly purified proteins. For E. coli DNA replication, the major initiator protein (purple), the helicase (yellow), and the primase (blue) are the dnaA, dnaB, and dnaG proteins, respectively. In the first step, many molecules of the initiator protein bind to specific DNA sequences at the replication origin and destabilize the double helix by forming a filamentous structure in which the DNA is wrapped around the protein. Next, two helicases are brought in by helicase-loading proteins (the dnaC proteins; brown), which inhibit the helicases until they are properly loaded at the replication origin. (The helicase-loading proteins prevent the replicative DNA helices from inappropriately entering other singlestrand stretches of DNA in the bacterial genome.) Aided by single-strand binding protein (not shown), the loaded helicases further separate the DNA strands, thereby enabling primases to enter and synthesize initial primers. In subsequent steps, two complete replication forks are assembled at the origin and move in opposite directions away from the replication origin. The initiator proteins are displaced as the lefthand fork moves through them.
图 5-26 在细菌中启动 DNA 复制的蛋白质。所示机制是通过体外使用高度纯化的蛋白质混合物进行研究建立的。对于大肠杆菌 DNA 复制,主要的启动蛋白(紫色)、解旋酶(黄色)和原始酶(蓝色)分别是 dnaA、dnaB 和 dnaG 蛋白。在第一步中,许多启动蛋白分子结合到复制起源处的特定 DNA 序列,并通过形成 DNA 缠绕在蛋白质周围的丝状结构来使双螺旋不稳定。接下来,两个解旋酶由解旋酶加载蛋白(dnaC 蛋白;棕色)带入,这些蛋白抑制解旋酶,直到它们在复制起源处正确加载为止。(解旋酶加载蛋白防止复制 DNA 螺旋不适当地进入细菌基因组中的其他单链 DNA 区段。)在单链结合蛋白的帮助下(未显示),加载的解旋酶进一步分离 DNA 链,从而使原始酶进入并合成初始引物。 在随后的步骤中,在起源处组装了两个完整的复制叉,它们朝着相反的方向远离复制起源。当左侧叉通过它们移动时,启动蛋白被排斥。
Figure 5-27 Methylation of the E. coli replication origin creates a refractory period for DNA initiation. DNA
图 5-27 大肠杆菌复制起源的甲基化会产生 DNA 起始的不可逾越期。DNA
methylation occurs at GATC sequences, 11 of which are found in the origin of replication (spanning approximately 250 nucleotide pairs). In its hemimethylated state (that is, one strand of the DNA methylated, the other unmethylated), the origin of replication is bound by an inhibitor protein (Seq A, not shown), which blocks the ability of the initiator proteins to unwind the origin DNA. About 15 minutes after replication is initiated, the hemimethylated origins become fully methylated by a DNA methylase enzyme; Seq A then dissociates allowing the origin of replication to become active.
甲基化发生在 GATC 序列上,其中有 11 个位于复制起始点(跨越大约 250 个核苷酸对)。在其半甲基化状态下(即 DNA 的一条链甲基化,另一条链未甲基化),复制起始点被一个抑制蛋白(未显示的 Seq A)结合,阻止启动蛋白展开起始点 DNA 的能力。在复制启动大约 15 分钟后,半甲基化的起始点会被 DNA 甲基转移酶完全甲基化;然后 Seq A 解离,使复制起始点变得活跃。
A single enzyme, the Dam methylase, is responsible for methylating all E. coli GATC sequences. As discussed earlier in the chapter, a lag in methylation after the replication of GATC sequences is also used by the E. coli mismatch proofreading system to distinguish the newly synthesized DNA strand from the parent DNA strand; in that case, the relevant GATC sequences are scattered throughout the chromosome, and they are not bound by Seq A.
一种酶,Dam 甲基化酶,负责甲基化所有大肠杆菌 GATC 序列。正如本章前面讨论的那样,在 GATC 序列复制后甲基化的滞后也被大肠杆菌错配校对系统用来区分新合成的 DNA 链与母体 DNA 链;在这种情况下,相关的 GATC 序列分散在整个染色体上,并且它们不受 Seq A 的约束。

in culture are labeled for a short time with -thymidine so that the DNA synthesized during this period becomes highly radioactive. The cells are then gently lysed, and the DNA is stretched on the surface of a glass slide coated with a photographic emulsion. Development of the emulsion in the dark reveals the pattern of labeled DNA through a technique known as autoradiography. The time allotted for radioactive labeling is chosen to allow each replication fork to move several micrometers along the DNA, so that the replicated DNA can be detected in the light microscope as lines of silver grains (radioactivity exposes photographic emulsion much as light does), even though the DNA molecule itself is too thin to be visible. In this way, both the rate and the direction of replication-fork movement can be determined (Figure 5-28). From the rate at which tracks of replicated DNA increase in length with increasing labeling time, the eukaryotic replication forks are estimated to travel at about 50 nucleotides per second. This is approximately twentyfold slower than the rate at which bacterial replication forks move, possibly reflecting the increased difficulty of replicating DNA that is packaged in chromatin.
在培养基中,细胞被标记了一段时间,使用 -胸腺嘧啶,使得在此期间合成的 DNA 变得高度放射性。然后轻柔地裂解细胞,将 DNA 拉伸到涂有感光乳剂的玻璃载玻片表面上。在黑暗中显影感光乳剂,通过一种称为放射自显影的技术揭示标记 DNA 的模式。选择放射性标记的时间允许每个复制叉沿 DNA 移动数微米,以便在光学显微镜中检测到复制的 DNA,表现为银粒线(放射性暴露感光乳剂,就像光线一样),即使 DNA 分子本身太细无法看见。通过这种方式,可以确定复制叉移动的速率和方向(图 5-28)。通过随着标记时间增加而增加的复制 DNA 轨迹长度的速率,估计真核生物复制叉每秒大约移动 50 个核苷酸。 这大约比细菌复制叉移动的速度慢了二十倍,可能反映了在染色质中包装的 DNA 复制的困难增加。
An average-size human chromosome contains a single linear DNA molecule of about 150 million nucleotide pairs. It would take 0.02 seconds/nucleotide nucleotides seconds (about 35 days) to replicate such a DNA molecule from end to end with a single replication fork moving at a rate of 50 nucleotides per second. As expected, therefore, the autoradiographic experiments just described reveal that many forks, belonging to separate replication bubbles, are moving simultaneously on each eukaryotic chromosome.
一个平均大小的人类染色体包含大约 1.5 亿个核苷酸对的单一线性 DNA 分子。如果以每秒 50 个核苷酸的速度移动的单个复制叉从一端到另一端复制这样一条 DNA 分子,需要 0.02 秒/核苷酸,即 1.5 亿个核苷酸需要 3,000,000 秒(约 35 天)。因此,正如预期的那样,刚刚描述的放射自显影实验揭示了许多复制叉,属于不同的复制泡泡,在每个真核染色体上同时移动。
Much more sophisticated methods now exist for monitoring DNA replication initiation and tracking the movement of DNA replication forks across whole genomes. If a population of cells can be synchronized so they all begin DNA replication at the same time, the amount of each segment of DNA in the genome can be determined at specific time points using one of the DNA sequencing methods described in Chapter 8. Because a segment of a genome that has been replicated will contain twice as much DNA as an unreplicated segment, replication-fork initiation and fork movement can be accurately monitored across an entire genome.
目前存在更加复杂的方法来监测 DNA 复制起始并跟踪 DNA 复制叉在整个基因组中的移动。如果一群细胞可以被同步化,使它们同时开始 DNA 复制,那么可以使用第 8 章中描述的 DNA 测序方法之一,在特定时间点确定基因组中每个 DNA 片段的数量。因为已经复制的基因组片段将包含两倍于未复制片段的 DNA 量,所以可以准确监测整个基因组中的复制叉起始和叉移动。
Experiments of this type have shown the following: (1) Approximately 30,00050,000 origins of replication are used each time a human cell divides. (2) The human genome has many more (perhaps tenfold more) potential origins than this, and different cell types use different sets of origins. This excess of origins may allow a cell to coordinate its active origins with other features of its chromosomes such as which genes are being expressed. The excess origins also provide "backups" in case a primary origin fails. (3) Origins of replication do not all "fire" simultaneously; rather, they often are activated in a prescribed order in a given cell type. (4) Regardless of when a given origin fires or where on the chromosome it is located, the replication forks all move at approximately the same speed. (5) As in bacteria, replication forks are formed in pairs and create an expanding
这类实验显示了以下结果:(1)每次人类细胞分裂时大约使用 30,000 至 50,000 个复制起点。(2)人类基因组可能有更多(可能是十倍以上)的潜在起点,不同的细胞类型使用不同的起点集。这种过剩的起点可能使细胞能够将其活跃的起点与其染色体的其他特征协调,比如哪些基因正在表达。过剩的起点还提供了“备用”,以防主要起点失败。(3)复制起点并非全部同时“启动”;相反,在给定细胞类型中,它们通常按照规定的顺序被激活。(4)无论给定起点何时启动或其在染色体上的位置如何,复制叉都以大致相同的速度移动。(5)与细菌一样,复制叉成对形成并创建一个扩展。
Figure 5-28 The experiments that first demonstrated the pattern in which replication forks are formed and move on eukaryotic chromosomes. The new DNA made in human cells in culture was labeled briefly with a pulse of highly radioactive thymidine -thymidine). (A) In this experiment, the cells were lysed, and the DNA was stretched out on a glass slide that was subsequently covered with a photographic emulsion. After several months, the emulsion was developed, revealing a line of silver grains over the radioactive DNA. The brown DNA in this figure is shown only to help with the interpretation of the autoradiograph; the unlabeled DNA is invisible in such experiments. (B) This experiment was the same except that a further incubation in unlabeled medium allowed additional DNA, with a lower level of radioactivity, to be replicated. The pairs of dark tracks in B were found to have silver grains tapering off in opposite directions, demonstrating bidirectional fork movement from a central replication origin where a replication bubble forms (see Figure 5-24). A replication fork is thought to stop only when it encounters a replication fork moving in the opposite direction or when it reaches the end of the chromosome; in this way, all the DNA is eventually replicated.
图 5-28 首次展示了复制叉在真核染色体上形成并移动的模式的实验。在培养的人类细胞中,新合成的 DNA 被短暂地标记为高度放射性的胸腺嘧啶脉冲 -胸腺嘧啶)。(A)在这个实验中,细胞被裂解,DNA 被拉直放在玻璃片上,随后覆盖上一层感光乳剂。几个月后,乳剂显影,显示出一行银颗粒在放射性 DNA 上。这幅图中的棕色 DNA 仅用于帮助解释自显影片;在这类实验中,未标记的 DNA 是看不见的。(B)这个实验与 A 相同,只是在未标记培养基中进一步孵育,使得额外的 DNA 以较低放射性水平复制。B 中的暗色轨迹成对出现,银颗粒朝相反方向逐渐减少,证明了双向叉运动,从一个中央复制起源点开始,形成一个复制泡泡(见图 5-24)。 复制叉被认为只有在遇到相反方向移动的复制叉或到达染色体末端时才会停止;通过这种方式,最终所有的 DNA 都会被复制。

replication bubble as they move in opposite directions away from a common point of origin, stopping only when they meet a replication fork moving in the opposite direction or when they reach a chromosome end. In this way, many replication forks operate independently on each chromosome and yet form two complete daughter DNA helices.
当它们沿着相反方向从一个共同起点移动时,复制泡会停止,只有当它们遇到相反方向移动的复制叉或到达染色体末端时才会停止。这样,许多复制叉在每条染色体上独立运作,却形成两条完整的子 DNA 螺旋。

In Eukaryotes, DNA Replication Takes Place During Only One Part of the Cell Cycle
在真核生物中,DNA 复制只发生在细胞周期的某个阶段

When growing rapidly, bacteria replicate their DNA nearly continually. In contrast, DNA replication in most eukaryotic cells occurs only during a specific part of the cell-division cycle, called the DNA synthesis phase, or S phase (Figure 5-29). In a mammalian cell, the S phase typically lasts for about 8 hours; in simpler eukaryotic cells such as yeasts, the phase can be as short as 40 minutes. By its end, each chromosome has been replicated to produce two complete copies, which remain joined together at their centromeres until the M phase ( for mitosis), which soon follows. Although different origins of replication fire at different times, all DNA replication is begun and completed during S phase. In Chapter 17, we describe the control system that runs the cell cycle, and we explain how entry into each phase of the cycle requires the cell to have successfully completed the previous phase.
当细菌快速生长时,它们几乎持续复制其 DNA。相比之下,大多数真核细胞中的 DNA 复制仅发生在细胞分裂周期的特定阶段,称为 DNA 合成阶段或 S 期(图 5-29)。在哺乳动物细胞中,S 期通常持续约 8 小时;在较简单的真核细胞如酵母中,S 期可能只有 40 分钟。到 S 期结束时,每条染色体都已复制成两份完整的拷贝,在其着丝粒处保持连接,直到随后的 M 期(有丝分裂期)。尽管不同的复制起点在不同时间点启动,但所有 DNA 复制都在 S 期开始并完成。在第 17 章中,我们描述了负责细胞周期的控制系统,并解释了进入周期的每个阶段都要求细胞成功完成前一个阶段。
In the following sections, we explore how DNA replication begins on eukaryotic chromosomes and how this event is coordinated with the cell cycle.
在接下来的章节中,我们将探讨 DNA 复制如何在真核染色体上开始,以及这一事件如何与细胞周期协调。

Eukaryotic Origins of Replication Are "Licensed" for Replication by the Assembly of an Origin Recognition Complex

Having seen that a eukaryotic chromosome is replicated using many origins of replication, each of which fires at a characteristic time in S phase of the cell cycle, we turn to the nature of these origins of replication. We saw earlier in this chapter that replication origins have been precisely defined in bacteria as specific DNA sequences that attract initiator proteins, which then assemble the DNA replication machinery. We shall see that this is also the case for the singlecell budding yeast . cerevisiae, but it appears not to be strictly true for many other eukaryotes.
在看到真核染色体是使用许多复制起源进行复制的,每个起源在细胞周期的 S 期以特定时间发生后,我们转向这些复制起源的性质。我们在本章前面看到,复制起源在细菌中已被精确定义为特定的 DNA 序列,吸引启动蛋白,然后组装 DNA 复制机器。我们将看到,在单细胞酵母 . cerevisiae 中也是如此,但对许多其他真核生物来说似乎并非严格如此。
For budding yeast, the location of every origin of replication on each chromosome has been determined. The particular chromosome shown in Figure 5-30-chromosome III from S. cerevisiae-is one of the smallest chromosomes known, with a length less than that of a typical human chromosome. Its major origins are spaced an average of 30,000 nucleotide pairs apart, but only a subset of these origins is used by a given cell. Nonetheless, this chromosome can be replicated in about 15 minutes.
对于酿酒酵母,已确定了每条染色体上每个复制起点的位置。在图 5-30 中显示的特定染色体-来自酿酒酵母的染色体 III-是已知最小的染色体之一,长度不到典型人类染色体的 。其主要起点平均间隔 30,000 个核苷酸对,但只有给定细胞使用其中的一个子集。尽管如此,这条染色体大约可以在 15 分钟内复制。
The minimal DNA sequence required for directing DNA replication initiation in S. cerevisiae has been determined by taking a segment of DNA that spans an origin of replication and testing smaller and smaller DNA fragments for their ability to function as origins. These DNA sequences that can serve as an origin of replication are found to contain (1) a binding site for a large, multisubunit initiator protein called ORC, for origin recognition complex; (2) a stretch of DNA that is rich in A's and T's and therefore easy to pull apart; and (3) at least one binding site for proteins that facilitate ORC binding, probably by adjusting the local chromatin structure.
在酿酒酵母中确定了引导 DNA 复制起始所需的最小 DNA 序列,方法是取跨越复制起始点的 DNA 片段,测试越来越小的 DNA 片段是否能作为起始点发挥功能。这些能够作为复制起始点的 DNA 序列被发现包含(1)一个用于大型多亚基启动蛋白 ORC(起始点识别复合物)的结合位点;(2)一个富含 A 和 T 的 DNA 区段,因此易于分离;以及(3)至少一个蛋白结合位点,用于促进 ORC 结合,可能通过调整局部染色质结构。
nucleotide pairs (thousands)
Figure 5-29 The four successive phases of a standard eukaryotic cell cycle. During the , and phases, the cell grows continually. During phase growth stops, the nucleus divides, and the cell divides in two. DNA replication is confined to the part of the cell cycle known as phase. is the gap between phase and phase; is the gap between phase and phase. Many eukaryotic cells spend only a small fraction of their time in phase.
图 5-29 标准真核细胞周期的四个连续阶段。在 阶段,细胞持续生长。在 阶段,生长停止,细胞核分裂,细胞分裂成两个。DNA 复制仅限于被称为 阶段的细胞周期部分。 阶段和 阶段之间的间隙; 阶段和 阶段之间的间隙。许多真核细胞只在 阶段花费很少的时间。
Figure 5-30 The origins of DNA replication on chromosome III of the yeast S. cerevisiae. This chromosome, one of the smallest eukaryotic chromosomes known, carries a total of 180 genes. As indicated, it contains 18 replication origins, although they are used with different frequencies. Those in red are typically used in less than of cell divisions, while those in green are used about of the time.
图 5-30 酿酒酵母 S. cerevisiae 染色体 III 上 DNA 复制的起源。这个染色体是已知的最小的真核染色体之一,携带了总共 180 个基因。如图所示,它包含 18 个复制起源,尽管它们的使用频率不同。红色表示的通常在 的细胞分裂中使用,而绿色表示的大约在 的时间中使用。

Features of the Human Genome That Specify Origins of Replication Remain to Be Fully Understood

Compared with the situation in budding yeast, the determinants of replication origins in humans have been difficult to discover. It has been possible to identify specific human DNA sequences, each several thousand nucleotide pairs in length, that are sufficient to serve as replication origins. These origins continue to function when moved to a different chromosomal region by recombinant DNA methods, as long as they are placed in a region where the chromatin is relatively uncondensed. However, comparisons of such DNA sequences have not revealed DNA sequences in common as in the origins of bacteria and yeasts.
与酿酒酵母相比,人类复制起源的决定因素很难发现。已经能够识别出特定的人类 DNA 序列,每个序列长达数千个核苷酸对,足以作为复制起源。这些起源在通过重组 DNA 方法移动到不同染色体区域时继续发挥功能,只要它们被放置在染色质相对不致密的区域。然而,对这些 DNA 序列的比较并未揭示出类似细菌和酵母起源中常见的 DNA 序列。
Despite this, a human ORC that is very similar to the yeast ORC binds to origins of replication and initiates DNA replication in humans. Many of the other proteins that function in the initiation process in yeast likewise have central roles in humans. The yeast and human initiation mechanisms are thus similar, although some property of the genome other than a specific DNA sequence has the central role in attracting an ORC to a mammalian origin of replication. Origins of replication are often nucleosome-free, and it has been proposed that DNA that is difficult to fold onto a histone core may help define origins of replication. Nearby transcriptional activity on the genome may also play a role in activating certain origins, by altering the local chromatin structures, as we discuss in Chapter 7. This idea helps to explain why different cell types-which express different sets of genes-often use different origins. Consistent with this idea, origins that fire the earliest in S phase tend to be located near highly transcribed regions of the genome.
尽管如此,与酵母 ORC 非常相似的人类 ORC 结合到复制起源并在人类中启动 DNA 复制。在酵母中参与启动过程的许多其他蛋白质同样在人类中发挥核心作用。因此,酵母和人类的启动机制是相似的,尽管基因组的某些特性除了特定的 DNA 序列之外,在吸引 ORC 到哺乳动物复制起源中起着核心作用。复制起源通常是核小体自由的,有人提出 DNA 难以折叠到组蛋白核心上可能有助于定义复制起源。基因组上附近的转录活动也可能通过改变局部染色质结构在激活某些起源中发挥作用,正如我们在第 7 章中讨论的那样。这个想法有助于解释为什么不同的细胞类型-表达不同基因组合-通常使用不同的起源。与这个想法一致的是,在 S 期中最早发生的起源往往位于基因组高度转录区域附近。
Finally, origins located in proximity to each other tend to fire together, and it seems likely that the three-dimensional structure of chromosomes organizes origins of replication into domains, such that all the origins in a given domain fire simultaneously. All of these influences probably work together to determine how mammalian origins of replication are selected by the cell, thereby explaining the difficulty scientists have had in precisely defining their salient features.

Properties of the ORC Ensure That Each Region of the DNA Is Replicated Once and Only Once in Each S Phase
ORC 的特性确保 DNA 的每个区域在每个 S 期间仅被复制一次

In bacteria, once the initiator protein is properly bound to the single origin of replication, the assembly of the replication forks seems to follow more or less automatically. In eukaryotes, the situation is significantly different because of a profound problem eukaryotes have in replicating chromosomes: with so many places to begin replication, how is the process regulated to ensure that all the DNA is copied once and only once?
在细菌中,一旦启动蛋白正确结合到单个复制起点,复制叉的组装似乎更多或更少地自动进行。在真核生物中,情况显著不同,因为真核生物在复制染色体方面存在一个深刻问题:由于有这么多开始复制的地方,如何调节这个过程以确保所有 DNA 只复制一次?
The answer lies in how the assembly of the replication-fork protein at the origins of replication is regulated. We discuss this process in more detail in Chapter 17, where we consider the machinery that underlies the cell-division cycle. In brief, during phase, a symmetrical complex of two incomplete helicases is loaded onto DNA by the bound ORC. Then, upon passage from phase to phase, specialized protein kinases come into play and direct the final assembly of the two replicative helicases, positioning one on each of the two complementary DNA single strands, where they move in opposite directions to begin opening the DNA double helix. At this point, the additional replication proteins are brought to the DNA, and two complete replication forks move in opposite directions away from the origin of replication (Figure 5-31).
答案在于复制叉蛋白在复制起点的组装是如何调控的。我们在第 17 章中更详细地讨论了这个过程,其中我们考虑了支持细胞分裂周期的机制。简而言之,在 相期间,两个不完整解旋酶的对称复合物由结合的 ORC 加载到 DNA 上。然后,在从 相到 相的过程中,专门的蛋白激酶开始发挥作用,指导两个复制解旋酶的最终组装,将其定位在两个互补的 DNA 单链上,它们以相反方向移动,开始打开 DNA 双螺旋。在这一点上,额外的复制蛋白被带到 DNA 上,两个完整的复制叉朝着远离复制起点的相反方向移动(图 5-31)。
The same protein kinases that trigger the final assembly of the helicases prevent the binding of new helicases to that origin until the next phase resets the entire cycle (for details, see pp. 1043-1045). They do this, in part, by phosphorylating ORC, rendering it unable to accept new helicases. Thus, the kinases specify a single window of opportunity for precursor helicases to be loaded at origins of replication ( phase, when kinase activity is low) and a second window for
同样的蛋白激酶触发螺旋酶的最终组装,阻止新的螺旋酶结合到该起源,直到下一个 相重置整个周期(有关详细信息,请参见第 1043-1045 页)。它们部分地通过磷酸化 ORC 来实现这一点,使其无法接受新的螺旋酶。因此,激酶指定了一个单一的机会窗口,用于在复制起源处加载前体螺旋酶( 相,当激酶活性较低时),以及第二个窗口。
Figure 5-31 DNA replication initiation in eukaryotes. This mechanism ensures that each origin of replication is activated only once per cell cycle. An origin of replication can be used only if two Mcm helicases (which form the enzymatic cores of the replicative helicases) are loaded in phase. At the beginning of phase, specialized kinases phosphorylate both the Mcm helicases and ORC, activating the former and inactivating the latter. These kinases also guide the assembly of additional proteins that complete the helicases to form the fully active replicative helicases, known as the CMG helicases. New Mcm helicases cannot be loaded at the origin until the cell progresses through mitosis to the next phase, when ORC is dephosphorylated. The name CMG derives from Cdc45, Mcm, and GINS, the components of the active helicase (see Figure 5-19).
图 5-31 真核生物 DNA 复制起始。这种机制确保每个复制起始点在每个细胞周期中只被激活一次。只有在 相加载了两个 Mcm 解旋酶(形成复制解旋酶的酶心)后,才能使用复制起始点。在 相初期,专门的激酶磷酸化 Mcm 解旋酶和 ORC,激活前者并使后者失活。这些激酶还引导额外蛋白质的组装,完成解旋酶形成完全活跃的复制解旋酶,即 CMG 解旋酶。直到细胞通过有丝分裂进展到下一个 相时,新的 Mcm 解旋酶才能加载到起始点,此时 ORC 被去磷酸化。CMG 的名称来源于 Cdc45、Mcm 和 GINS,这是活跃解旋酶的组成部分(见图 5-19)。

them to be assembled into their active form (S phase, when kinase activity is high). Because these two phases of the cell cycle are mutually exclusive and occur in a prescribed order, each origin of replication can fire only once during each cell cycle.
它们被组装成它们的活性形式(S 期,激酶活性高时)。由于细胞周期的这两个阶段是互斥的,并按照规定的顺序发生,因此每个复制起点在每个细胞周期中只能发生一次。
Because there are many more potential replication origins on a eukaryotic chromosome than are actually used in any one cell cycle (see Figure 5-30), the DNA at many ORC-bound replication origins will be replicated by forks formed at a neighboring region of the chromosome. Thus, preventing any single origin from firing more than once during an phase is not enough to avoid the re-replication of DNA in eukaryotes. In addition, any ORC-DNA complex that is passed by a replication fork must be inactivated, and it is the combination of the two mechanisms that guarantees that each region of the DNA is replicated once and only once in each phase.
由于真核染色体上存在的潜在复制起始点比任何一个细胞周期中实际使用的要多得多(见图 5-30),许多 ORC 结合的复制起始点上的 DNA 将由在染色体相邻区域形成的叉形结构复制。因此,在 相期间阻止任何单个起始点多次发射并不足以避免真核生物中 DNA 的再复制。此外,任何被复制叉结构通过的 ORC-DNA 复合物必须被失活,而正是这两种机制的结合确保了 DNA 的每个区域在每个 相期间只被复制一次。

New Nucleosomes Are Assembled Behind the Replication Fork

Several additional aspects of DNA replication are specific to eukaryotes compared with bacteria. As discussed in Chapter 4, eukaryotic chromosomes are composed of roughly equal mixtures of DNA and protein. Chromosome duplication therefore requires not only the replication of DNA but also the synthesis of new chromosomal proteins and their assembly onto the DNA behind each replication fork. Although we are far from understanding this process in detail, we are beginning to learn how the fundamental unit of chromatin packaging, the nucleosome, is duplicated. The cell requires a large amount of new histone protein, approximately equal in mass to the newly synthesized DNA, each time it divides. For this reason, most eukaryotic organisms possess multiple copies of the gene for each histone. Vertebrate cells, for example, have about 20 repeated gene sets, most containing the genes that encode all five histones , , and ).
DNA 复制的几个额外方面与细菌相比,对真核生物具有特异性。正如第 4 章所讨论的,真核染色体由 DNA 和蛋白质大致相等的混合物组成。因此,染色体复制不仅需要复制 DNA,还需要合成新的染色体蛋白质并将其组装到每个复制叉后面的 DNA 上。尽管我们远未详细了解这一过程,但我们开始了解染色质包装的基本单位——核小体是如何复制的。每次细胞分裂时,细胞需要大量新的组蛋白蛋白质,其质量大致相等于新合成的 DNA。因此,大多数真核生物体拥有每种组蛋白的多个基因副本。例如,脊椎动物细胞大约有 20 个重复的基因组,其中大多数包含编码所有五种组蛋白的基因。
Unlike most proteins, which are made continually, histones are synthesized mainly in S phase, when the level of histone mRNA increases about fiftyfold as a result of both increased transcription and decreased mRNA degradation. The major histone mRNAs are degraded within minutes when DNA synthesis stops at the end of S phase. The mechanism depends on special properties of the ends of these mRNAs, as discussed in Chapter 7. In contrast to their mRNAs, the histone proteins themselves are remarkably stable and may survive for many generations. The tight linkage between DNA synthesis and histone synthesis appears to reflect a feedback mechanism that monitors the level of free histone to ensure that the amount of histone made exactly matches the amount of new DNA synthesized.
与大多数蛋白质不同,组蛋白主要在 S 期合成,当组蛋白 mRNA 水平增加约 50 倍时,这是由于转录增加和 mRNA 降解减少的结果。主要的组蛋白 mRNA 在 S 期结束时 DNA 合成停止后几分钟内被降解。该机制取决于这些 mRNA 的 端的特殊性质,如第 7 章所讨论的。与它们的 mRNA 相比,组蛋白蛋白质本身非常稳定,可以存活多代。DNA 合成和组蛋白合成之间的紧密联系似乎反映了一种反馈机制,监测游离组蛋白的水平,以确保制造的组蛋白数量与合成的新 DNA 数量完全匹配。
As a replication fork advances it must pass through the parent nucleosomes. In the cell, efficient replication requires chromatin remodeling complexes (discussed in Chapter 4) and histone chaperone proteins (discussed below) to destabilize the DNA-histone interfaces. Aided by such specialized proteins, replication forks can transit even highly condensed chromatin. As a replication fork passes through chromatin, the histones are transiently displaced leaving about 600 nucleotide pairs of "free" DNA in its wake. The reestablishment of nucleosomes behind a moving fork occurs in an intriguing way. When a nucleosome is traversed by a replication fork, the histone octamer is broken into an tetramer and two H2A-H2B dimers (discussed in Chapter 4), all of which are released from DNA. The H3-H4 tetramers remain in the vicinity of the fork by loosely binding to several of the proteins at the replication fork (primarily the CMG helicase) and are distributed at random to one or the other daughter duplexes as the fork moves forward. In contrast, the H2A-H2B dimers are released completely from the fork and may diffuse to entirely different chromosomes. Freshly made tetramers are added to the newly synthesized DNA to fill in the "spaces," and H2A-H2B dimers-half of which are old and half new-are then added at random
随着复制叉的推进,它必须穿过母核小体。在细胞中,高效的复制需要染色质重塑复合物(在第 4 章讨论)和组蛋白伴侣蛋白(下文讨论)来 destablize DNA-组蛋白界面。在这些专门的蛋白质的帮助下,复制叉甚至可以穿过高度浓缩的染色质。当复制叉穿过染色质时,组蛋白会暂时被移开,留下大约 600 个核苷酸对的“自由” DNA。在移动叉后重新建立核小体的过程非常有趣。当核小体被复制叉穿过时,组蛋白八聚体被分解成一个 四聚体和两个 H2A-H2B 二聚体(在第 4 章讨论),所有这些都从 DNA 中释放出来。H3-H4 四聚体通过松散地结合到复制叉处的几种蛋白质(主要是 CMG 解旋酶)而留在叉附近,并随着叉的前进随机分布到一个或另一个子代双链。相比之下,H2A-H2B 二聚体完全从叉中释放出来,可能扩散到完全不同的染色体。新制备的 四聚体被添加到新合成的 DNA 中,以填补“空隙”,然后 H2A-H2B 二聚体-其中一半是旧的,一半是新的-随机添加
to complete the nucleosomes behind the fork (Figure 5-32). The formation of new nucleosomes behind a replication fork has an important consequence for the process of DNA replication itself. As DNA polymerase discontinuously synthesizes the lagging strand (see Figure 5-19), the length of each Okazaki fragment is determined by the point at which DNA polymerase is blocked by a newly formed nucleosome. This tight coupling between nucleosome duplication and DNA replication probably explains why the length of Okazaki fragments in eukaryotes ( 200 nucleotides) is approximately the same as the nucleosome repeat length.
完成叉后的核小体(图 5-32)。在复制叉后形成新的核小体对 DNA 复制过程本身有重要影响。随着 DNA 聚合酶 不连续合成滞后链(见图 5-19),每个岡崎片段的长度由 DNA 聚合酶 被新形成的核小体阻断的点决定。核小体复制与 DNA 复制之间的紧密耦合可能解释了为什么真核生物中岡崎片段的长度(200 核苷酸)大致与核小体重复长度相同。
The orderly and rapid addition of new tetramers and dimers behind a replication fork requires histone chaperones (also called chromatin assembly factors). These multisubunit complexes bind the highly basic histones and release them on DNA only in the appropriate context. For example, some of the histone chaperones, along with their histone cargoes, are directed to newly replicated DNA through a specific interaction with the sliding clamp (see Figure 5-32). As we have seen, these clamps remain on the DNA behind replication forks, and some appear to linger just long enough for the histone chaperones to complete their tasks. Because they bind so well to histones, some histone chaperones also help to disassemble nucleosomes. Of particular importance to DNA replication is the FACT chaperone, which moves at the front of the replication machinery, disassembling nucleosomes as it moves forward (see Figure 5-32).
有序且迅速地在复制叉后添加新的 四聚体和 二聚体需要组蛋白伴侣(也称为染色质装配因子)。这些多亚基复合物结合高度碱性的组蛋白,并仅在适当的情境下释放它们在 DNA 上。例如,一些组蛋白伴侣与它们的组蛋白载体一起,通过与滑动夹具的特定相互作用被引导到新复制的 DNA 上(见图 5-32)。正如我们所见,这些夹具保留在复制叉后的 DNA 上,有些似乎停留的时间刚好足够组蛋白伴侣完成它们的任务。由于它们与组蛋白结合得很好,一些组蛋白伴侣还有助于解体核小体。对 DNA 复制特别重要的是 FACT 伴侣,它在复制机械的前面移动,随着前进解体核小体(见图 5-32)。

Termination of DNA Replication Occurs Through the Ordered Disassembly of the Replication Fork
DNA 复制的终止是通过有序拆卸复制叉进行的

We saw earlier in this chapter that E. coli DNA replication begins at a single origin, and two replication forks proceed bidirectionally around the circular genome, meeting at a spot opposite to the origin of replication. Here, the two forks do not simply collide with each other running at full speed; rather, this spot on the E. coli genome has a special DNA sequence that slows down and stalls the movement of each fork, causing them to disassemble. The remaining gaps in the daughter DNA molecules are filled in and sealed by repair DNA polymerases and DNA ligase (see Figures 5-11 and 5-12), and the two completed bacterial genomes are separated using topoisomerases (see Figure 5-23).
我们在本章前面看到,大肠杆菌 DNA 复制始于一个单一起源,两个复制叉以双向方式绕着圆形基因组前进,最终在与复制起源相对的位置相遇。在这里,这两个复制叉并不是简单地以全速相撞;相反,大肠杆菌基因组上的这个位置有一个特殊的 DNA 序列,可以减慢并使每个叉停滞不前,导致它们解体。子 DNA 分子中剩余的缺口由修复 DNA 聚合酶和 DNA 连接酶填补并封闭(见图 5-11 和 5-12),然后使用拓扑异构酶分离两个完成的细菌基因组(见图 5-23)。
As might be expected, the situation in eukaryotes is more complicated. First, each round of replication requires many termination events, roughly as many as there are initiation events at origins of replication. Thus, in mammalian cells, approximately termination events occur in every S phase. Second, the termination of replication forks in eukaryotes is largely independent of any underlying DNA sequence in the genome. Rather, the principal termination
正如人们所预料的那样,在真核生物中的情况更为复杂。首先,每一轮复制都需要许多终止事件,大致与复制起始点的启动事件数量相同。因此,在哺乳动物细胞中,每个 S 期大约会发生 次终止事件。其次,在真核生物中,复制叉的终止在很大程度上独立于基因组中的任何 DNA 序列。相反,主要的终止机制

Figure 5-32 Formation of nucleosomes behind a replication fork. Parent tetramers remain associated with the fork and are distributed at random to the daughter DNA molecules, with roughly equal numbers inherited by each daughter. In contrast, dimers are released completely from the fork as it passes. This release begins just in front of the replication fork and is facilitated by the histone chaperone FACT, which moves with the fork. FACT has several globular protein domains connected by flexible linkers and can make multiple contacts with a nucleosome to aid in its disassembly. Additional histone chaperones (NAP1 and CAF1) restore the full complement of histones to daughter molecules using both parent and newly synthesized histones. Although not shown in the figure, it has been proposed that FACT directly hands off parent tetramers to components of the replication machinery, which in turn hand them off to CAF1 chaperones, which deposit them evenly on the two daughter molecules. The way in which histones are distributed behind a replication fork means that some daughter nucleosomes contain only parent histones or only newly synthesized histones, but most are hybrids of old and new. For simplicity, the DNA double helix is shown as a single red line.
图 5-32 复制叉后面核小体的形成。母 四聚体保持与叉结合,并随机分布到子 DNA 分子上,每个子分子继承的数量大致相等。相反, 二聚体在叉通过时完全释放。这种释放始于复制叉的正前方,并由与叉一起移动的组蛋白伴侣 FACT 促进。FACT 具有几个由柔性连接器连接的球状蛋白结构域,可以与核小体多次接触,帮助其解体。额外的组蛋白伴侣(NAP1 和 CAF1)使用母本和新合成的组蛋白将完整的组蛋白补充到子分子中。尽管图中未显示,但有人提出 FACT 直接将母 四聚体移交给复制机械的组分,然后再将它们移交给 CAF1 组蛋白,后者将它们均匀地沉积在两个子分子上。 组蛋白在复制叉后面的分布方式意味着一些子核小体只包含母本组蛋白或新合成的组蛋白,但大多数是新旧组蛋白的混合体。为简单起见,DNA 双螺旋被表示为单一的红线。

signal is a head-on encounter with a fork moving in the opposite direction. When two forks meet, the CMG helicase at each fork is covalently modified by addition of ubiquitin (see Figure 3-65), which causes its disassembly and removal from DNA. Without the helicase, the other replication proteins rapidly dissociate from the fork. Repair DNA polymerase and DNA ligase subsequently fill in and seal any remaining gaps. Eukaryotic replication forks must also contend with the ends of chromosomes. Here, it is believed that the CMG helicase simply slides off the end of the DNA molecule, leading to the dissociation of the other fork proteins. However, replicating DNA to the very end of a chromosome presents a special challenge to the eukaryotic cell, as we describe next.
信号是与朝向相反方向移动的叉头对冲。当两个叉头相遇时,每个叉头上的 CMG 解旋酶会通过泛素的加成发生共价修饰(见图 3-65),导致其解体并从 DNA 中移除。没有解旋酶,其他复制蛋白会迅速从叉头中解离。修复 DNA 聚合酶和 DNA 连接酶随后填补并封闭任何剩余的缺口。真核复制叉头还必须应对染色体的末端。在这里,人们认为 CMG 解旋酶会简单地从 DNA 分子的末端滑落下来,导致其他叉头蛋白的解离。然而,将 DNA 复制到染色体的末端对真核细胞提出了特殊挑战,我们将在接下来的部分描述。

Telomerase Replicates the Ends of Chromosomes

We saw earlier in the chapter that synthesis of the lagging strand at a replication fork must occur discontinuously through a backstitching mechanism that produces short DNA fragments attached to RNA primers. The final RNA primer synthesized on the lagging-strand template cannot be replaced by DNA because there is no primer ahead of it to provide a end for the repair polymerase. Without a mechanism to deal with this problem, DNA would be lost from the ends of all chromosomes each time a cell divides.
我们在本章前面看到,在复制叉中,滞后链的合成必须通过一个反向缝合机制不连续地进行,这个机制产生与 RNA 引物连接的短 DNA 片段。在滞后链模板上合成的最终 RNA 引物不能被 DNA 替换,因为在它前面没有引物来为修复聚合酶提供一个 端。如果没有处理这个问题的机制,每次细胞分裂时,所有染色体的末端都会丢失 DNA。
Bacteria avoid this "end-replication" problem by having circular DNA molecules as chromosomes, as we have seen. Eukaryotes solve it in a different way: they have specialized nucleotide sequences at the ends of their chromosomes that are incorporated into structures called telomeres (discussed in Chapter 4). Telomeres contain many tandem repeats of a short sequence that is similar in organisms as diverse as protozoa, fungi, plants, and mammals. In humans, the sequence of the repeat unit is GGGTTA, and it is repeated roughly a thousand times at each telomere.
细菌通过拥有环状 DNA 分子作为染色体来避免这种“末端复制”问题,正如我们所见。真核生物以一种不同的方式解决这个问题:它们在染色体末端有专门的核苷酸序列,这些序列被整合到称为端粒的结构中(在第 4 章中讨论)。端粒包含许多短序列的串联重复,这些序列在原生动物、真菌、植物和哺乳动物等不同生物体中相似。在人类中,重复单元的序列是 GGGTTA,每个端粒大约重复一千次。
Telomere DNA sequences are recognized by sequence-specific DNA-binding proteins that attract an enzyme, called telomerase, that replenishes these sequences each time a cell divides. Telomerase recognizes the tip of an existing telomere DNA repeat sequence and elongates it in the -to- direction, using an RNA template that is a component of the enzyme itself to synthesize new DNA copies of the repeat (Figure 5-33). The enzymatic portion of telomerase resembles other reverse transcriptases, proteins that synthesize DNA using an RNA template, although, in this case, the telomerase RNA also contributes to the active site and is essential for efficient catalysis. After extension of the parent DNA strand by telomerase, replication of the lagging strand at the chromosome end can be completed by the conventional DNA polymerases, using these extensions as a template to synthesize the complementary strand (Figure 5-34).
端粒 DNA 序列被特异性 DNA 结合蛋白识别,这些蛋白吸引一种称为端粒酶的酶,该酶在每次细胞分裂时补充这些序列。端粒酶识别现有端粒 DNA 重复序列的末端,并在 - 方向上延长它,使用作为酶本身组成部分的 RNA 模板合成重复的新 DNA 拷贝(图 5-33)。端粒酶的酶部分类似于其他反转录酶,这些蛋白使用 RNA 模板合成 DNA,尽管在这种情况下,端粒酶 RNA 也对活性位点起作用,并且对于高效催化是必不可少的。在端粒酶延长母 DNA 链后,染色体末端的滞后链的复制可以由常规 DNA 聚合酶完成,使用这些延伸作为模板合成互补链(图 5-34)。
Figure 5-33 Schematic structure of human telomerase. This large enzyme is composed of 10 protein subunits and an RNA of 451 nucleotides. The RNA forms the scaffold of the complex, provides the template for synthesizing new DNA telomere repeats, and helps form the active site. The synthesis reaction itself is carried out by the reverse transcriptase domain of the protein, shown in light green, in conjunction with the RNA. A reverse transcriptase is a special form of polymerase enzyme that uses an RNA template to make a DNA strand; telomerase is unique in carrying its own RNA template with it. Telomerase also contains several additional protein complexes (some of which are shown in dark green and blue) that are needed to assemble the enzyme and, for many organisms but not humans, to bring it to the ends of chromosomes. (Modified from T.H.D. Nguyen et al., Nature 557: .
图 5-33 人类端粒酶的示意结构。这种大型酶由 10 个蛋白亚基和一个 451 个核苷酸的 RNA 组成。RNA 形成了复合物的支架,为合成新的 DNA 端粒重复序列提供模板,并帮助形成活性位点。合成反应本身由蛋白质的反转录酶结构域(浅绿色显示)与 RNA 一起进行。反转录酶是一种特殊形式的聚合酶酶,它使用 RNA 模板制造 DNA 链;端粒酶在携带其自身的 RNA 模板方面是独特的。端粒酶还包含几个额外的蛋白质复合物(其中一些显示为深绿色和蓝色),这些复合物需要组装酶,并且对许多生物体而言,但不包括人类,将其带到染色体的末端。(修改自 T.H.D. Nguyen 等人,自然 557:
Figure 5-34 Telomere replication. Shown here is the reaction that synthesizes the repeating sequences that form the ends of the chromosomes (telomeres) of eukaryotes. The 3' end of the parent lagging-strand template is extended by RNA-templated DNA synthesis; this allows the incomplete daughter DNA strand that is paired with it to be synthesized to the end of the chromosome. The synthesis of the final bit of lagging strand is carried out by DNA polymerase , which carries a DNA primase as one of its subunits (Movie 5.6). DNA polymerase is the same enzyme used to begin the synthesis of each Okazaki fragment on the lagging strand; it begins its synthesis with RNA (not shown) and continues with DNA (green). The telomere sequence illustrated is that of the ciliate Tetrahymena, in which these reactions were first discovered.
图 5-34 端粒复制。这里显示的是合成形成真核生物染色体末端(端粒)的重复序列的反应。母链滞后链模板的 3'端通过 RNA 模板 DNA 合成进行延伸;这使得与之配对的不完整的子 DNA 链能够合成至染色体末端。滞后链的最后一小部分的合成由 DNA 聚合酶 执行,该酶携带 DNA 引物酶作为其亚基之一(影片 5.6)。DNA 聚合酶 是用于在滞后链上开始每个 Okazaki 片段合成的相同酶;它以 RNA(未显示)开始合成,然后继续合成 DNA(绿色)。所示的端粒序列是被首次发现这些反应的纤毛虫类 Tetrahymena 的序列。

Telomeres Are Packaged into Specialized Structures That Protect the Ends of Chromosomes

The ends of chromosomes present cells with an additional problem. As we will see in the next part of this chapter, when a chromosome is accidently broken into two pieces, the break is rapidly repaired. Telomeres must clearly be distinguished from these accidental breaks; otherwise, the cell will attempt to "repair" telomeres, generating chromosome fusions and other genetic abnormalities. Telomeres have several features to prevent this from happening.
A specialized nuclease chews back the end of a telomere leaving a protruding, single-strand 3' end. This protruding end-in combination with the GGGTTA repeats in telomeres-attracts a group of proteins that form a protective chromosome cap known as shelterin. In particular, shelterin protects telomeres from being treated as damaged DNA. Another feature of telomeres may offer additional protection. When human telomeres are artificially cross-linked and viewed by electron microscopy, structures known as "t-loops" can be observed in which the protruding single-strand end of the telomere loops back and tucks itself into the duplex DNA of the telomere repeat sequence (Figure 5-35). An attractive idea is that t-loops are orchestrated by shelterin to help "hide" the very ends of chromosomes.
一种专门的核酸酶咬掉端粒的 端,留下一个突出的、单链的 3'端。这个突出的端与端粒中的 GGGTTA 重复序列结合,吸引一组蛋白质形成一种被称为 shelterin 的保护染色体帽。特别是,shelterin 保护端粒免受被视为受损 DNA 的处理。端粒的另一个特征可能提供额外的保护。当人类端粒被人为交联并通过电子显微镜观察时,可以观察到称为“t-环”的结构,其中端粒的突出单链末端回环并塞入端粒重复序列的双链 DNA 中(图 5-35)。一个吸引人的想法是,t-环由 shelterin 协调以帮助“隐藏”染色体的末端。

Telomere Length Is Regulated by Cells and Organisms

Because the processes that grow and shrink each telomere sequence are only approximately balanced, chromosome ends contain variable numbers of telomeric repeats. Not surprisingly, many cells, including stem cells and germ cells, have homeostatic mechanisms that maintain the number of these repeats within a limited range (Figure 5-36).
由于增长和缩短每个端粒序列的过程仅大致平衡,染色体末端包含可变数量的端粒重复序列。毫不奇怪,许多细胞,包括干细胞和生殖细胞,具有维持这些重复序列数量在有限范围内的稳态机制(图 5-36)。
In most of the dividing somatic cells of humans, however, telomeres gradually shorten, and it has been proposed that this provides a counting mechanism that helps prevent the unlimited proliferation of wayward cells in adult tissues. In its simplest form, this idea holds that our somatic cells start off in the embryo with a full complement of telomeric repeats. These are then eroded to different extents in different cell types. Some stem cells, notably those in tissues that must be replenished at a high rate throughout life-bone marrow or gut lining, for example-retain full telomerase activity. However, in many other types of cells, the level of telomerase is reduced so that the enzyme cannot quite keep up with chromosome duplication. Such cells lose 100-200 nucleotides from each telomere every time they divide. After many cell generations, the descendant cells will inherit chromosomes that lack functioning telomeres, and, as a result of this defect, activate a DNA-damage response causing them to withdraw permanently from the cell cycle and cease dividing-a process called replicative cell senescence (discussed in Chapters 17 and 20). In theory, such a mechanism could provide a
在人类大多数体细胞的分裂过程中,端粒逐渐缩短,有人提出这提供了一种计数机制,有助于防止成体组织中不受控制的细胞无限增殖。简单来说,这个想法认为我们的体细胞在胚胎中以完整的端粒重复序列开始。然后,这些序列在不同的细胞类型中被侵蚀到不同程度。一些干细胞,特别是那些在整个生命过程中必须高速再生的组织中的干细胞,例如骨髓或肠道内膜,保留了完整的端粒酶活性。然而,在许多其他类型的细胞中,端粒酶的水平降低,以至于酶无法跟上染色体复制的速度。这些细胞每次分裂时会从每个端粒失去 100-200 个核苷酸。经过多代细胞分裂后,后代细胞将继承缺乏功能性端粒的染色体,并且由于这种缺陷,激活 DNA 损伤反应,导致它们永久退出细胞周期并停止分裂-这个过程称为复制细胞衰老(在第 17 章和第 20 章中讨论)。理论上,这样的机制可以提供一种

Figure 5-35 A t-loop at the end of a mammalian chromosome. (A) Electron micrograph of the DNA at the end of an interphase human chromosome. The chromosome was fixed, deproteinated, and artificially thickened before viewing. The loop seen here is approximately 15,000 nucleotide pairs in length. (B) Schematic diagram of t-loop formation. (A, from J.D. Griffith et al., Cell 97:503-514, 1999. With permission from Elsevier.)
图 5-35 哺乳动物染色体末端的 t-环。(A) 人类染色体间期末端 DNA 的电子显微镜图。在观察之前,染色体被固定、去蛋白化并人为增厚。这里看到的环大约有 15,000 个核苷酸对的长度。(B) t-环形成的示意图。(A, 参见 J.D. Griffith 等人,Cell 97:503-514, 1999. 获 Elsevier 许可。)
telomere repeats 端粒重复
safeguard against the uncontrolled cell proliferation of abnormal cells in somatic tissues, thereby helping to protect us from cancer.
The idea that telomere length acts as a "measuring stick" to count cell divisions and thereby regulate the lifetime of the cell lineage has been tested in several ways. For certain types of human cells grown in tissue culture, the experimental results support such a theory. Human fibroblasts normally proliferate for about 60 cell divisions in culture before undergoing replicative cell senescence. Like most other somatic cells in humans, fibroblasts produce only low levels of telomerase, and their telomeres gradually shorten each time they divide. When telomerase is provided to the fibroblasts by inserting a fully active telomerase gene, telomere length is maintained and many of the cells now continue to proliferate indefinitely. Also consistent with these ideas is the observation that, in approximately of cancer cells, the telomerase gene has become reactivated, thereby circumventing the normal safety mechanism (see pp. 1073-1074).
端粒长度作为“计数细胞分裂次数并从而调节细胞谱系寿命”的概念已经通过多种方式进行了测试。对于在组织培养中培养的某些类型的人类细胞,实验结果支持这样的理论。人类成纤维细胞在培养中通常进行大约 60 次细胞分裂后就会经历复制性细胞衰老。与人类其他多数体细胞一样,成纤维细胞只产生很低水平的端粒酶,它们的端粒在每次分裂时逐渐缩短。当通过插入一个完全活跃的端粒酶基因向成纤维细胞提供端粒酶时,端粒长度得以维持,许多细胞现在继续无限增殖。与这些想法一致的还有这样的观察结果,即在大约 的癌细胞中,端粒酶基因已经重新激活,从而规避了正常的安全机制(见 1073-1074 页)。
It has been proposed that this type of control on cell proliferation may contribute to the aging of animals like ourselves. These ideas have been tested by producing transgenic mice that lack telomerase entirely. The telomeres in mouse chromosomes are about five times longer than human telomeres, and the mice must therefore be bred through three or more generations before their telomeres have shrunk to the normal human length. It is therefore perhaps not surprising that the first generations of mice develop normally. However, the mice in later generations develop progressively more defects in some of their highly proliferative tissues. In addition, these mice show signs of premature aging and have a pronounced tendency to develop tumors. In these and other respects, these mice resemble humans with the genetic disease dyskeratosis congenita. Individuals afflicted with this disease carry one functional and one nonfunctional copy of the telomerase RNA gene; they have prematurely shortened telomeres and typically die of progressive bone marrow failure. These individuals also develop lung scarring and liver cirrhosis and show abnormalities in various epidermal structures including skin, hair follicles, and nails.
已经提出,这种对细胞增殖的控制可能会导致像我们这样的动物的衰老。通过制造完全缺乏端粒酶的转基因小鼠来测试这些想法。小鼠染色体上的端粒大约比人类端粒长五倍,因此这些小鼠必须经过三代或更多代的繁殖,直到它们的端粒缩短到正常人类长度为止。因此,也许并不奇怪,第一代小鼠会正常发育。然而,后代小鼠在一些高度增殖的组织中逐渐出现更多缺陷。此外,这些小鼠显示出早衰的迹象,并且有明显的肿瘤发展倾向。在这些方面和其他方面,这些小鼠类似于患有遗传疾病角化异常症的人类。患有这种疾病的个体携带一个功能性和一个非功能性的端粒酶 RNA 基因拷贝;他们的端粒会过早缩短,通常会死于进行性骨髓衰竭。 这些个体还会发展肺部瘢痕和肝硬化,并显示出各种表皮结构异常,包括皮肤、毛囊和指甲。
The above observations demonstrate that controlling cell proliferation by telomere shortening poses a risk to an organism, because not all of the cells that begin losing the ends of their chromosomes will stop dividing. Some apparently become genetically unstable, but continue to divide, giving rise to variant cells that can lead to cancer. As discussed above, many of these variant cells
Figure 5-36 A demonstration that yeast cells control the length of their telomeres. In this experiment, the telomere at one end of a particular chromosome is artificially made either longer (left) or shorter (right) than average. After many cell divisions, the chromosome recovers, showing an average telomere length and a length distribution that is typical of the other chromosomes in the yeast cell. A similar feedback mechanism for controlling telomere length has been proposed for the germ-line cells and stem cells of mammals.
图 5-36 证明酵母细胞控制其端粒长度的实验。在这个实验中,某一染色体的一端的端粒被人为地延长(左)或缩短(右)至平均长度之外。经过多次细胞分裂后,该染色体恢复,显示出平均端粒长度和长度分布,与酵母细胞中其他染色体典型的情况相似。类似的反馈机制被提出用于控制哺乳动物的生殖细胞和干细胞的端粒长度。

ultimately produce high levels of telomerase, thereby ensuring their continued survival. Clearly, the use of telomere shortening as a regulating mechanism is not foolproof and, like many mechanisms in the cell, it must strike a balance between benefit and risk.

Summary 摘要

The proteins that initiate DNA replication bind to DNA sequences at a replication origin to catalyze the formation of a replication bubble with two outward-moving replication forks. The process begins when an initiator protein-DNA complex is formed that subsequently loads a DNA helicase onto the DNA template. Other proteins are then added to form the multienzyme "replication machine" that catalyzes DNA synthesis at each replication fork.
启动 DNA 复制的蛋白质结合到复制起源处的 DNA 序列上,催化形成具有两个向外移动的复制叉的复制泡泡。该过程始于形成一个启动蛋白质-DNA 复合物,随后将 DNA 解旋酶加载到 DNA 模板上。然后添加其他蛋白质以形成多酶“复制机器”,在每个复制叉处催化 DNA 合成。
In bacteria and some simple eukaryotes, replication origins are defined by specific DNA sequences that are several hundred nucleotide pairs long. In other eukaryotes, such as humans, features that specify an origin of DNA replication are less well defined, and probably depend more on structural features of chromosomes than on specific DNA sequences.
在细菌和一些简单的真核生物中,复制起源由几百个核苷酸对长的特定 DNA 序列定义。在其他真核生物中,如人类,指定 DNA 复制起源的特征定义不太明确,可能更依赖于染色体的结构特征而不是特定的 DNA 序列。
Bacteria typically have a single origin of replication in a circular chromosome. With fork speeds of up to 1000 nucleotides per second, they can replicate their genome in less than an hour. Eukaryotic DNA replication takes place in only one part of the cell cycle, the S phase. The replication fork in eukaryotes moves about 20 times more slowly than the bacterial replication fork, and the much longer eukaryotic chromosomes each require many replication origins to complete their replication in an S phase, which typically lasts for 8 hours in human cells. The different replication origins in these eukaryotic chromosomes are activated in a sequence, determined in part by which genes are currently being transcribed and the structure of chromatin across each chromosome. After the replication fork has passed, chromatin structure is re-formed by the addition of new histones to the old histones that are directly inherited by each daughter DNA molecule.
细菌通常在圆形染色体中有一个复制起点。以每秒 1000 个核苷酸的速度,它们可以在不到一个小时内复制其基因组。真核 DNA 复制仅发生在细胞周期的一个部分,即 S 期。真核生物的复制叉移动速度大约比细菌复制叉慢 20 倍,而更长的真核染色体每个都需要许多复制起点才能在 S 期内完成复制,人类细胞中 S 期通常持续 8 小时。这些真核染色体中的不同复制起点按顺序激活,部分取决于当前正在转录的基因以及每个染色体上染色质的结构。复制叉通过后,染色质结构通过向每个子 DNA 分子直接继承的旧组蛋白添加新组蛋白重新形成。
Eukaryotes solve the problem of replicating the ends of their linear chromosomes with a specialized end structure, the telomere, maintained by a special nucleotidepolymerizing enzyme called telomerase. Telomerase extends one of the DNA strands at the end of a chromosome by using an RNA template that is an integral part of the enzyme itself, producing a highly repeated DNA sequence that typically extends for thousands of nucleotide pairs at each chromosome end. Telomeres have specialized structures that distinguish them from broken ends of chromosomes, ensuring that they are not treated as damaged DNA.
真核生物通过一种特殊的末端结构——端粒来解决线性染色体末端复制的问题,端粒由一种称为端粒酶的特殊核苷酸聚合酶维持。端粒酶利用其内在的 RNA 模板延伸染色体末端的 DNA 链之一,产生一个高度重复的 DNA 序列,通常在每个染色体末端延伸数千个核苷酸对。端粒具有特殊的结构,使其与染色体断裂末端有所区别,确保它们不被视为受损的 DNA。


Maintaining the genetic stability that an organism needs for its survival requires not only an extremely accurate mechanism for replicating DNA but also mechanisms for repairing the many accidental lesions that DNA continually suffers. Most such spontaneous changes in DNA are temporary because they are immediately corrected by a set of processes that are collectively called DNA repair. Of the tens of thousands of random changes created every day in the DNA of a human cell by heat, metabolic accidents, radiation of various sorts, and exposure to substances in the environment, only a few (less than ) accumulate as permanent mutations in the DNA sequence. The rest are eliminated with remarkable efficiency by DNA repair.
维持生物体生存所需的遗传稳定性不仅需要一种极其精确的 DNA 复制机制,还需要修复 DNA 持续遭受的许多意外损伤的机制。DNA 中的大多数自发变化是暂时的,因为它们会立即被一组被称为 DNA 修复的过程所纠正。在人类细胞的 DNA 中,每天由热量、代谢意外、各种辐射以及环境中物质的暴露造成的成千上万的随机变化中,只有少数(小于 )会积累为 DNA 序列中的永久突变。其余的则会被 DNA 修复以非凡的效率消除。
The importance of DNA repair is evident from the large investment that cells make in the enzymes that carry it out: several percent of the coding capacity of most genomes is devoted solely to DNA repair functions. The importance of DNA repair is also demonstrated by the increased rate of mutation that follows the inactivation of a DNA repair gene. Many DNA repair proteins and the genes that encode them-which we now know operate in a wide range of organisms,
DNA 修复的重要性显而易见,细胞在进行 DNA 修复时投入了大量资源:大多数基因组的编码能力中有几个百分点专门用于 DNA 修复功能。DNA 修复的重要性还体现在 DNA 修复基因失活后突变率的增加。许多 DNA 修复蛋白质及其编码基因,我们现在知道它们在广泛的生物体中发挥作用。
TABLE 5-2 Some Inherited Human Syndromes with Defects in DNA Repair
表 5-2 一些具有 DNA 修复缺陷的遗传性人类综合征

Name of syndrome or
responsible genes
Phenotype 表型 Enzyme or process affected
Msh2, Msh3, Msh6, Mlh1, Pms2
Colon cancer 结肠癌 Mismatch repair 不匹配修复

Polymerase proofreading-
associated polyposis
Colon cancer 结肠癌 Proofreading by DNA polymerase
DNA 聚合酶的校对
Aicardi-Goutières syndrome

Encephalopathy, neurological dysfunction,
genome instability

DNA 中错误插入的核糖核苷酸的去除
Removal of misincorporated ribonucleotides
in DNA

Xeroderma pigmentosum (XP) A-G 组
Xeroderma pigmentosum (XP)
groups A-G

Skin cancer, UV sensitivity, neurological
Nucleotide excision repair
Cockayne syndrome 库克恩综合征 UV sensitivity, developmental abnormalities
UV 敏感性,发育异常

Coupling of nucleotide excision repair to
XP variant XP 变体 UV sensitivity, skin cancer
UV 敏感性,皮肤癌
Translesion synthesis by DNA polymerase
DNA 聚合酶 的转录延伸
Ataxia telangiectasia (AT)

白血病,淋巴瘤, -射线敏感性,基因组不稳定性
Leukemia, lymphoma, -ray sensitivity,
genome instability

ATM 蛋白,一种被双链 DNA 断裂激活的蛋白激酶
ATM protein, a protein kinase activated by
double-strand DNA breaks
Seckel syndrome 西克尔综合征 Dwarfism, microcephaly 侏儒症,小头畸形

ATR 蛋白,一种被单链 DNA 断裂激活的蛋白激酶
ATR protein, a protein kinase activated by
single-strand DNA breaks
Brca1 Brca1 Brca1 Breast and ovarian cancer
Repair by homologous recombination
Brca2 Brca2 Brca2

Breast, ovarian, prostate, and pancreatic
Repair by homologous recombination

disorder (ATLD)

白血病,淋巴瘤, -射线敏感性,基因组不稳定性
Leukemia, Iymphoma, -ray sensitivity,
genome instability

Mre11 蛋白,用于处理双链 DNA 断裂
Mre11 protein, required for processing
double-strand DNA breaks
Werner syndrome 沃纳氏综合征

Premature aging, cancer at several sites,
genome instability

辅助 3'-外切酶和 DNA 解旋酶用于修复
Accessory 3 '-exonuclease and DNA
helicase used in repair
Bloom syndrome 布鲁姆综合征

Cancer at several sites, stunted growth,
genome instability
DNA helicase needed for recombination
需要重组的 DNA 解旋酶
Fanconi anemia groups A-W
范可尼贫血 A-W 组

Congenital abnormalities, leukemia, genome
DNA interstrand cross-link repair
DNA 互链交联修复
46BR patient 46BR 患者

对 DNA 损伤剂的高敏感性,基因组不稳定性
Hypersensitivity to DNA-damaging agents,
genome instability
DNA ligase I DNA 连接酶 I
including humans-were originally identified in bacteria by the isolation and characterization of mutants that displayed an increased mutation rate or an increased sensitivity to DNA-damaging agents.
包括人类在内的生物最初是通过分离和表征显示出增加突变率或对 DNA 损伤剂显示出增加敏感性的突变体来鉴定的。
Studies of the consequences of a diminished capacity for DNA repair in humans have linked many human diseases with decreased repair (Table 5-2). Thus, we saw previously that defects in a human gene whose product normally functions to repair the mismatched base pairs resulting from DNA replication errors can lead to an inherited predisposition to cancers of the colon and some other organs, caused by an increased mutation rate. In another human disease, xeroderma pigmentosum (XP), the afflicted individuals have an extreme sensitivity to ultraviolet radiation because they are unable to repair the damage to DNA caused by this component of sunlight. This repair defect results in an increased mutation rate that leads to serious skin lesions and a greatly increased susceptibility to skin cancers. Finally, mutations in the Brcal and Brca2 genes compromise a type of DNA repair known as homologous recombination and are a major cause of hereditary breast and ovarian cancers.
研究表明,人类 DNA 修复能力降低会导致许多人类疾病与修复能力下降相关(Table 5-2)。因此,我们之前发现,人类基因缺陷会导致遗传性易患结肠癌和其他一些器官癌症,这是由于 DNA 复制错误导致的错配碱基对无法修复,从而导致突变率增加。在另一种人类疾病——黑色素瘤性干皮病(XP)中,患者对紫外线极度敏感,因为他们无法修复受阳光中紫外线引起的 DNA 损伤。这种修复缺陷导致突变率增加,进而导致严重的皮肤病变和极大的皮肤癌易感性增加。最后,Brcal 和 Brca2 基因的突变会影响一种称为同源重组的 DNA 修复类型,并且是遗传性乳腺癌和卵巢癌的主要原因。

Without DNA Repair, Spontaneous DNA Damage Would Rapidly Change DNA Sequences
没有 DNA 修复,自发性 DNA 损伤会迅速改变 DNA 序列

Although DNA is a highly stable material—as required for the storage of genetic information-it is a complex organic molecule that is susceptible, even under normal cell conditions, to spontaneous changes that would lead to mutations if left unrepaired (Figure 5-37 and see Table 5-3). For example, the DNA of each human cell loses about 18,000 purine bases (adenine and guanine) every day because their -glycosyl linkages to deoxyribose break, a spontaneous hydrolysis reaction called depurination. Similarly, a spontaneous deamination of cytosine to uracil in
尽管 DNA 是一种高度稳定的材料——这是存储遗传信息所必需的,但它是一种复杂的有机分子,即使在正常细胞条件下也容易受到影响,如果不进行修复,就会导致突变(图 5-37 和参见表 5-3)。例如,每个人类细胞的 DNA 每天会失去约 18,000 个嘌呤碱基(腺嘌呤和鸟嘌呤),因为它们的 -脱氧核糖链与脱氧核糖断裂,这是一种自发的水解反应,称为去嘌呤作用。同样,胞嘧啶自发地脱氨基转变为尿嘧啶。

Figure 5-37 A summary of spontaneous alterations that require DNA repair. The sites on each nucleotide modified by spontaneous oxidative damage (red arrows), hydrolytic attack (blue arrows), and methylation (green arrows) are shown, with the width of each arrow indicating the relative frequency of each event (see Table 5-3). (After T. Lindahl, Nature 362:709-715, 1993.)
图 5-37 显示需要 DNA 修复的自发改变摘要。显示了每个核苷酸上由自发氧化损伤(红色箭头)、水解攻击(蓝色箭头)和甲基化(绿色箭头)修改的位点,每个事件的相对频率由每个箭头的宽度表示(参见表 5-3)。 (摘自 T. Lindahl, Nature 362:709-715, 1993 年。)

TABLE 5-3 Endogenous DNA Lesions Arising and Repaired in a Diploid Mammalian Cell in 24 Hours
表 5-3 24 小时内在二倍体哺乳动物细胞中产生和修复的内源性 DNA 损伤
DNA lesion DNA 损伤 Number repaired in   中的数字已修复
Hydrolysis 水解
Depurination 去嘌呤 18,000
Depyrimidination 去嘧啶化 600
Cytosine deamination 胞嘧啶脱氨基化 100
5-Methylcytosine deamination
Oxidation 氧化
8-oxoguanine 8-氧鸟嘌呤 1500

Ring-saturated pyrimidines (thymine glycol, cytosine

Lipid peroxidation products (M1G, etheno-A,
Nonenzymatic methylation by S-adenosylmethionine
7-Methylguanine 7-甲基鸟嘌呤 6000
3-Methyladenine 3-甲基腺嘌呤 1200
Nonenzymatic methylation by nitrosated polyamines and peptides
-Methylguanine  - 甲基鸟嘌呤

表中列出的 DNA 损伤是细胞内正常化学反应的结果。暴露于外部化学物质和辐射的细胞遭受更严重和更多样化的 DNA 损伤。
The DNA lesions listed in the table are the result of the normal chemical reactions that take
place in cells. Cells that are exposed to external chemicals and radiation suffer greater and more
diverse forms of DNA damage. (From T. Lindahl and D.E. Barnes, Cold Spring Harb. Symp.
Quant. Biol. 65:127-133, 2000.)
DNA strand DNA 链
DNA strand DNA 链
DNA occurs at a rate of about 100 bases per cell per day (Figure 5-38). DNA bases are also occasionally damaged by encounters with reactive metabolites produced in the cell (for example, the high-energy methyl donor, -adenosylmethionine) or by exposure to toxic chemicals in the environment. Likewise, ultraviolet radiation from the Sun can produce a covalent linkage between two adjacent pyrimidine bases in DNA to form, for example, thymine dimers (Figure 5-39). If left uncorrected, most of these changes would lead either to the deletion of one or more base pairs or to a base-pair substitution in the daughter DNA chain when the DNA is replicated (Figure 5-40). These mutations would then be propagated throughout all subsequent cell generations. Such a high rate of unrepaired random changes in the DNA sequence would have disastrous consequences, both in the germ line and in somatic tissues.
DNA 每天以大约 100 个碱基的速率发生(图 5-38)。DNA 碱基有时也会受到与细胞内产生的反应性代谢产物(例如,高能量的甲基供体, -腺甲硫氨酸)的接触或暴露于环境中的有毒化学物质的损害。同样,来自太阳的紫外辐射可以在 DNA 中的两个相邻嘧啶碱基之间形成共价键连接,例如,胸腺嘧啶二聚体(图 5-39)。如果不加以纠正,这些变化中的大多数将导致一个或多个碱基对的缺失,或者在 DNA 复制时导致子 DNA 链中的碱基对替换(图 5-40)。然后这些突变将在所有后续细胞代中传播。这种高速率的未修复 DNA 序列中的随机变化将在生殖细胞系和体细胞组织中产生灾难性后果。
DNA strand DNA 链
Figure 5-39 The ultraviolet radiation in sunlight can cause the formation of thymine dimers. Two adjacent thymine bases have become covalently attached to each other to form a thymine dimer. Skin cells that are exposed to sunlight are especially susceptible to this type of DNA damage. Dimers can also form between an adjacent thymine and cytosine.
图 5-39 太阳光中的紫外线辐射会导致胸腺嘧啶二聚体的形成。两个相邻的胸腺嘧啶碱基已经共价结合在一起形成了一个胸腺嘧啶二聚体。暴露在阳光下的皮肤细胞特别容易受到这种类型的 DNA 损伤。二聚体也可以在相邻的胸腺嘧啶和胞嘧啶之间形成。

Figure 5-38 Depurination and deamination are the most frequent spontaneous chemical reactions known to create serious DNA damage in cells. (A) Depurination can remove guanine (or adenine) from DNA. (B) The major type of deamination reaction converts cytosine to uracil, which, as we have seen, is not normally found in DNA. However, deamination can occur on other bases as well. Both depurination and deamination take place on double-helical DNA, and neither reaction breaks the phosphodiester backbone.
图 5-38 脱嘌呤和脱氨是已知在细胞中造成严重 DNA 损伤的最常见的自发化学反应。(A) 脱嘌呤可以从 DNA 中去除鸟嘌呤(或腺嘌呤)。(B) 脱氨的主要类型反应将胞嘧啶转化为尿嘧啶,正如我们所见,尿嘧啶在 DNA 中通常不会被发现。然而,脱氨也可能发生在其他碱基上。脱嘌呤和脱氨都发生在双螺旋 DNA 上,且两种反应均不会破坏磷酸二酯骨架。
Figure 5-40 Chemical modifications of nucleotides, if left unrepaired, produce mutations. (A) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. As shown in Figure 5-43, deamination of cytosine produces uracil. Uracil differs from cytosine in its basepairing properties and preferentially basepairs with adenine. The DNA replication machinery therefore inserts an adenine when it encounters a uracil on the template strand. (B) Depurination, if uncorrected, can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it can skip to the next complete nucleotide, as shown, thus producing a daughter DNA molecule that is missing one nucleotide pair. In other cases, the replication machinery places an incorrect nucleotide across from the missing base, again resulting in a mutation (not shown).
图 5-40 核苷酸的化学修饰,如果未修复,会产生突变。(A) 胞嘧啶的脱氨基,如果未纠正,会导致 DNA 复制时一个碱基被另一个替代。如图 5-43 所示,胞嘧啶的脱氨基会产生尿嘧啶。尿嘧啶在碱基配对性质上与胞嘧啶不同,并且更倾向于与腺嘌呤配对。因此,当 DNA 复制机构在模板链上遇到尿嘧啶时,会插入一个腺嘌呤。(B) 脱嘌呤,如果未纠正,可能导致一个核苷酸对的丢失。当复制机构在模板链上遇到缺失的嘌呤时,会跳过到下一个完整的核苷酸,如图所示,从而产生一个缺少一个核苷酸对的子 DNA 分子。在其他情况下,复制机构会在缺失碱基的对面放置一个不正确的核苷酸,再次导致突变(未显示)。
(B) 核苷酸切除修复
G A T G C C A G A T G A T A C C 基因 A T G C C A G A T G A T A C C
DNA helix with 12 nucleotide gap
DNA 螺旋带有 12 个核苷酸间隙
DNA 聚合酶利用底链作为模板添加新核苷酸;DNA 连接酶封闭断裂
Figure 5-41 A comparison of two major DNA repair pathways. (A) Base excision repair. This pathway starts with a DNA glycosylase. In the example shown here, the enzyme uracil DNA glycosylase removes an accidentally deaminated cytosine in DNA. After the action of this glycosylase (or another DNA glycosylase that recognizes a different kind of damage), the sugar phosphate with the missing base is cut out by the sequential action of AP endonuclease and a phosphodiesterase. The gap of a single nucleotide is then filled by DNA polymerase and DNA ligase. The net result is that the that was created by accidental deamination is restored to a C. The loss of a base can occur either from the actions of DNA glycosylases that recognize damaged bases or from spontaneous chemical reactions (see Figure 5-37). AP endonuclease is so named because it recognizes any site in the DNA helix that contains a deoxyribose sugar with a missing base; such sites can arise either by the loss of a purine (apurinic sites) or by the loss of a pyrimidine (apyrimidinic sites). (B) Nucleotide excision repair. In bacteria, after a multienzyme complex has recognized a lesion such as a pyrimidine dimer (see Figure 5-39), one cut is made on each side of the lesion, and an associated DNA helicase then removes the entire portion of the damaged strand. The excision repair machinery in bacteria operates as shown. In humans, once the damaged DNA is recognized, a helicase is recruited to locally unwind the DNA duplex. Next, the excision nuclease enters and cleaves on either side of the damage, leaving a gap of about 30 nucleotides that is subsequently filled in. The nucleotide excision repair machinery in both bacteria and humans can recognize and repair many different types of DNA damage.
图 5-41 两种主要 DNA 修复途径的比较。 (A)碱基切除修复。 该途径始于 DNA 醣苷酶。 在此处所示的示例中,鸟嘌呤 DNA 醣苷酶酶去除 DNA 中意外脱氨基胞嘧啶。 在这种醣苷酶的作用之后(或者识别不同类型损伤的另一种 DNA 醣苷酶的作用之后),缺失碱基的磷酸糖将被 AP 内切核酸酶和磷酸二酯酶的连续作用切除。 然后,DNA 聚合酶和 DNA 连接酶填补一个单核苷酸的间隙。 最终结果是,由意外脱氨基生成的 被恢复为 C。 碱基的丢失可以是由于识别受损碱基的 DNA 醣苷酶的作用,也可以是由于自发化学反应(见图 5-37)引起的。 AP 内切核酸酶之所以如此命名,是因为它识别 DNA 螺旋中包含缺失碱基的脱氧核糖糖的任何位点; 这种位点可以通过丢失嘌呤(无嘌呤位点)或丢失嘧啶(无嘧啶位点)而产生。 (B)核苷酸切除修复。 在细菌中,当一个多酶复合物识别到一个损伤,比如嘧啶二聚体(见图 5-39),在损伤的两侧各做一次切割,然后一个相关的 DNA 解旋酶移除整个受损链的部分。细菌中的切除修复机制如图所示运作。在人类中,一旦受损的 DNA 被识别,一个解旋酶被招募来局部展开 DNA 双链。接下来,切除核酸酶进入并在损伤的两侧切割,留下大约 30 个核苷酸的间隙,随后填补。细菌和人类中的核苷酸切除修复机制可以识别和修复许多不同类型的 DNA 损伤。
Figure 5-41A). Depurination, which is by far the most frequent type of damage suffered by DNA, also leaves a deoxyribose sugar with a missing base. Depurinations are directly repaired beginning with AP endonuclease, following the bottom half of the pathway in Figure 5-41A.
图 5-41A)。脱嘌呤是 DNA 遭受的最常见损伤类型,也会使去氧核糖糖分子缺失一个碱基。脱嘌呤损伤可以直接通过 AP 内切酶修复,遵循图 5-41A 中路径的下半部分开始修复。
The second major repair pathway is called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. Such "bulky lesions" include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcinogen benzopyrene, found in tobacco smoke, coal tar, and diesel exhaust), as well as the various pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. In this pathway, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of
第二个主要的修复途径被称为核苷酸切除修复。这种机制可以修复几乎任何 DNA 双螺旋结构发生较大变化造成的损伤。这种“体积庞大的损伤”包括由 DNA 碱基与大型碳氢化合物(如致癌物苯并芘,存在于烟草烟雾、煤焦油和柴油尾气中)共价反应产生的损伤,以及由阳光引起的各种嘧啶二聚体(T-T、T-C 和 C-C)。在这个途径中,一个大型多酶复合物扫描 DNA,寻找双螺旋结构的扭曲,而不是特定碱基的改变。一旦发现损伤,它会在异常链的两侧切割磷酸二酯骨架。
(B) the distortion, and a DNA helicase peels away the single-strand oligonucleotide containing the lesion. The large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase (see Figure 5-41B).
(B)扭曲,DNA 解旋酶剥离含有损伤的单链寡核苷酸。然后 DNA 聚合酶和 DNA 连接酶修复 DNA 螺旋中产生的大缺口(见图 5-41B)。
An alternative to these base and nucleotide excision repair processes is the direct chemical reversal of DNA damage, and this strategy is selectively employed for the rapid removal of certain highly mutagenic or cytotoxic lesions. For example, the lesion -methylguanine has its methyl group removed by direct transfer to a cysteine residue in the repair protein itself. Because the repair protein is destroyed in the process, each molecule of it can only be used once. In another example, methyl groups in the lesions 1-methyladenine and 3-methylcytosine are "burned off" by an iron-dependent demethylase, with release of formaldehyde from the methylated DNA and regeneration of the native base.
这些碱基和核苷酸切除修复过程的替代方案是直接化学逆转 DNA 损伤,这种策略被选择性地用于快速去除某些高度致突变或细胞毒性的损伤。例如,损伤 -甲基鸟嘌呤通过直接转移至修复蛋白质中的半胱氨酸残基而去除其甲基基团。由于修复蛋白质在过程中被破坏,因此每个分子只能使用一次。在另一个例子中,损伤 1-甲基腺嘌呤和 3-甲基胞嘧啶中的甲基基团通过依赖铁的去甲基酶“燃烧”,从甲基化 DNA 中释放甲醛并再生原始碱基。

Coupling Nucleotide Excision Repair to Transcription Ensures That the Cell's Most Important DNA Is Efficiently Repaired
将核苷酸切除修复与转录耦合,确保细胞中最重要的 DNA 得到高效修复

All of a cell's DNA is under constant surveillance for damage, and the repair mechanisms we have described act on all parts of the genome. However, cells have a way of directing DNA repair to the DNA sequences that are most needed. They do this by linking RNA polymerase, the enzyme that transcribes DNA into RNA as the first step in gene expression, to the nucleotide excision repair pathway. As discussed above, this repair system can correct many different types of DNA damage. RNA polymerase stalls at DNA lesions and, through the use of coupling proteins, directs the excision repair machinery to those sites, thereby selectively repairing genes that are in current use by the cell. In bacteria, where genes are relatively short, the stalled RNA polymerase can be dissociated from the DNA; the DNA is repaired, and the gene is transcribed again from the beginning. In eukaryotes, where genes can be enormously long, a more complex reaction is used to "back up" the RNA polymerase, repair the damage, and then restart the polymerase.
细胞的所有 DNA 都在不断监视损伤,我们所描述的修复机制作用于基因组的所有部分。然而,细胞有一种方法可以将 DNA 修复引导到最需要的 DNA 序列。它们通过将 RNA 聚合酶(一种将 DNA 转录为 RNA 的酶,是基因表达的第一步)与核苷酸切除修复途径联系起来来实现这一点。正如上文所讨论的,这种修复系统可以纠正许多不同类型的 DNA 损伤。RNA 聚合酶在 DNA 损伤处停滞,并通过耦合蛋白的使用,将切除修复机制引导到这些位置,从而有选择地修复细胞当前使用的基因。在细菌中,基因相对较短,停滞的 RNA 聚合酶可以与 DNA 解离;DNA 得到修复,基因再次从头开始转录。在真核生物中,基因可能非常长,使用更复杂的反应来“备份”RNA 聚合酶,修复损伤,然后重新启动聚合酶。
The importance of transcription-coupled excision repair is seen in people with Cockayne syndrome, which is caused by a defect in this coupling. These individuals suffer from growth retardation, skeletal abnormalities, progressive neural retardation, and severe sensitivity to sunlight. Most of these problems are thought to arise from RNA polymerase molecules that become permanently stalled at sites of DNA damage that lie in important genes.
转录耦合修复的重要性在患有科克恩综合征的人群中得以体现,该综合征是由于这种耦合的缺陷引起的。这些个体患有生长迟缓、骨骼异常、进行性神经迟缓和对阳光的严重敏感。这些问题大多被认为是由于 RNA 聚合酶分子在重要基因中的 DNA 损伤部位被永久阻塞而引起的。

The Chemistry of the DNA Bases Facilitates Damage Detection
DNA 碱基的化学性质有助于损伤检测

The DNA double helix is well suited for repair. As noted earlier, it contains a backup copy of all genetic information. Equally importantly, the nature of the four bases in DNA makes the distinction between undamaged and damaged
DNA 双螺旋结构非常适合修复。正如前面所指出的,它包含了所有遗传信息的备份副本。同样重要的是,DNA 中四种碱基的性质使得未受损和受损之间的区别变得明显。

Figure 5-42 The recognition of an unusual nucleotide in DNA by baseflipping. The DNA glycosylase family of enzymes recognizes inappropriate bases in DNA in the conformation shown. Each of these enzymes cleaves the glycosyl bond that connects a particular recognized base (yellow) to the backbone sugar, removing it from the DNA. (A) Stick model of the DNA; (B) space-filling model.
图 5-42 DNA 中不寻常核苷酸的识别通过翻转碱基。DNA 醣苷酶家族的酶识别 DNA 中不当碱基的构象如图所示。这些酶中的每一个都会切断连接特定识别碱基(黄色)与骨架糖的醣苷键,将其从 DNA 中移除。(A)DNA 的棍状模型;(B)填充模型。
adenine 腺嘌呤
guanine 鸟嘌呤
cytosine 胞嘧啶
thymine 胸腺嘧啶
hypoxanthine 次黄嘌呤
5-methylcytosine 5-甲基胞嘧啶
thymine 胸腺嘧啶
bases very clear. For example, every possible deamination event in DNA yields an "unnatural" base, which can be directly recognized and removed by a specific DNA glycosylase. Hypoxanthine, for example, is the simplest purine base capable of pairing specifically with C. But hypoxanthine is not used in DNA, presumably because it is the direct deamination product of A. Instead G, with a second amino group, pairs with : cannot form from by spontaneous deamination, and its own deamination product (xanthine) is likewise unique (Figure 5-43).
碱基非常清晰。例如,DNA 中的每次可能的脱氨基事件都会产生一种“非自然”碱基,可以被特定的 DNA 醣苷酶直接识别并去除。例如,次黄嘌呤是能够与 C 特异配对的最简单的嘌呤碱基。但是次黄嘌呤在 DNA 中并未被使用,可能是因为它是 A 的直接脱氨基产物。相反,带有第二个氨基基团的 G 与 配对: 不能通过自发脱氨基形成 ,其自身的脱氨基产物(黄嘌呤)同样独特(图 5-43)。

Figure 5-43 The deamination of DNA nucleotides. In each case, the oxygen atom that is added in this reaction with water is colored red. (A) The spontaneous deamination products of and are recognizable as unnatural when they occur in DNA and thus are readily found and repaired, as is the deamination of to ; has no amino group to remove. (B) About of the nucleotides in vertebrate DNAs are methylated to help in controlling gene expression (discussed in Chapter 7). When these 5-methyl C nucleotides are accidentally deaminated, they form the natural nucleotide T. This T will be paired with a G on the opposite strand, forming a mismatched base pair.
图 5-43 DNA 核苷酸的脱氨作用。在每种情况下,通过水反应添加的氧原子为红色。 (A)当 DNA 中发生 的自发脱氨产物时,这些产物是不自然的,因此很容易被发现和修复,就像 的脱氨一样; 没有氨基可去除。 (B)在脊椎动物 DNA 中,约 的核苷酸被甲基化,以帮助控制基因表达(在第 7 章讨论)。当这些 5-甲基 C 核苷酸被意外脱氨时,它们形成自然核苷酸 T。 这个 T 将与对侧链上的 G 配对,形成不匹配的碱基对。
As discussed in Chapter 6, RNA is thought, on an evolutionary time scale, to have served as the genetic material before DNA, and it seems likely that the genetic code was initially carried in the four nucleotides , and . This raises the question of why the in RNA was replaced in DNA by (which is 5 -methyl ). We have seen that the spontaneous deamination of converts it to , but that this event is rendered relatively harmless by uracil DNA glycosylase. However, if DNA contained as a natural base, the repair system would not be able to distinguish a deaminated from a naturally occurring .
正如第 6 章所讨论的,从进化的时间尺度来看,RNA 被认为在 DNA 之前作为遗传物质,并且很可能遗传密码最初是由四个核苷酸携带的。这引发了一个问题,即为什么 RNA 中的 被 DNA 中的 (即 5-甲基 )所取代。我们已经看到, 的自发脱氨作用将其转化为 ,但是尿嘧啶 DNA 糖苷酶使这一事件相对无害。然而,如果 DNA 含有 作为一种自然碱基,修复系统将无法区分脱氨的 和自然存在的
A special situation occurs in vertebrate DNA, in which selected C nucleotides are methylated at specific CG sequences that are associated with inactive genes (discussed in Chapter 7). The accidental deamination of these methylated C nucleotides produces the natural nucleotide T (see Figure 5-43B) in a mismatched base pair with a G on the opposite DNA strand. To help in repairing deaminated methylated C nucleotides, a special DNA glycosylase recognizes a mismatched base pair involving in the sequence and removes the . This DNA repair mechanism must be relatively ineffective, however, because methylated C nucleotides are exceptionally common sites for mutations in vertebrate DNA. It is striking that, even though only about of the nucleotides in human DNA are methylated, mutations in these methylated nucleotides account for about one-third of the single-base mutations that have been observed in inherited human diseases.
脊椎动物 DNA 中发生了一种特殊情况,即选择性地在与非活性基因相关的特定 CG 序列上甲基化了某些 C 核苷酸(见第 7 章讨论)。这些甲基化的 C 核苷酸的意外脱氨基会产生天然核苷酸 T(见图 5-43B),与对面 DNA 链上的 G 形成不匹配的碱基对。为了帮助修复脱氨基化的甲基化 C 核苷酸,一种特殊的 DNA 醣基酶识别涉及 在序列 中的不匹配碱基对并去除 。然而,这种 DNA 修复机制必须相对无效,因为在脊椎动物 DNA 中,甲基化的 C 核苷酸是异常常见的突变位点。令人惊讶的是,即使人类 DNA 中只有约 中的 核苷酸被甲基化,这些甲基化核苷酸的突变却占据了遗传性人类疾病中观察到的单碱基突变约三分之一。

Special Translesion DNA Polymerases Are Used in Emergencies
特殊的转录 DNA 聚合酶在紧急情况下被使用

If a cell's DNA suffers heavy damage, the repair mechanisms that we have discussed are often insufficient to cope with it. In these cases, a different strategy is called into play, one that entails some risk to the cell. The highly accurate replicative DNA polymerases stall when they encounter damaged DNA, and in emergencies cells employ versatile, but less accurate, backup polymerases, known as translesion polymerases, to replicate through the DNA damage.
如果细胞的 DNA 遭受严重损伤,我们讨论过的修复机制通常无法应对。在这些情况下,会采用一种不同的策略,这种策略对细胞存在一定风险。当高度精确的复制 DNA 聚合酶遇到损伤的 DNA 时会停滞,细胞在紧急情况下会利用多功能但准确性较低的备用聚合酶,即称为跨损伤聚合酶,来复制经过 DNA 损伤的区域。
Human cells contain seven different translesion polymerases, some of which can recognize a specific type of DNA damage and add the nucleotides required to restore the correct sequence. For example, one such polymerase adds two A's opposite a thymine dimer (see Figure 5-39). Others make only "good guesses," especially when the template base has been extensively damaged. These enzymes are not as accurate as the normal replicative polymerases even when they copy an undamaged DNA sequence. For one thing, they lack exonucleolytic proofreading activity; in addition, many are much less discriminating than the replicative polymerase in choosing which nucleotide to incorporate initially. Each such translesion polymerase is therefore given a chance to add only one or a few nucleotides before a high-fidelity replicative polymerase resumes DNA synthesis.
人类细胞含有七种不同的转录失效聚合酶,其中一些可以识别特定类型的 DNA 损伤,并添加所需的核苷酸以恢复正确的序列。例如,这样的一种聚合酶在嘧啶二聚体对面添加两个 A(见图 5-39)。其他的只能做出“良好的猜测”,特别是当模板碱基受到严重损伤时。即使复制未受损的 DNA 序列时,这些酶也不如正常的复制聚合酶准确。首先,它们缺乏外切核酸校对活性;此外,许多在最初选择要合并的核苷酸时比复制聚合酶要不那么具有区分性。因此,每种这样的转录失效聚合酶只有在高保真度的复制聚合酶恢复 DNA 合成之前才有机会添加一个或几个核苷酸。
Despite their usefulness in allowing heavily damaged DNA to be replicated, these translesion polymerases do, as noted above, pose risks to the cell. They are probably responsible for most of the base-substitution and single-nucleotide deletion mutations that accumulate in genomes. Not only do they frequently produce mutations when copying damaged DNA, they probably also generate mutationsat a low level-on undamaged DNA. Clearly, it is important for the cell to tightly regulate these polymerases, activating them only at sites of DNA damage. Exactly how this happens for each translesion polymerase remains to be discovered, but a conceptual model is presented in Figure 5-44. The same principle applies to many of the DNA repair processes discussed in this chapter: because the enzymes that carry out these reactions are potentially dangerous to the genome, they must be brought into play only at the appropriate damaged sites.
尽管这些转录失译聚合酶在允许严重受损的 DNA 进行复制方面非常有用,但正如上文所指出的,它们对细胞构成风险。它们很可能是导致基础替换和单核苷酸缺失突变在基因组中积累的主要原因。它们不仅在复制受损 DNA 时经常产生突变,而且很可能也在未受损的 DNA 上以低水平产生突变。显然,对于细胞来说,紧密调控这些聚合酶非常重要,只在 DNA 损伤部位激活它们。每种转录失译聚合酶如何实现这一点仍有待发现,但在图 5-44 中提出了一个概念模型。同样的原则也适用于本章讨论的许多 DNA 修复过程:因为执行这些反应的酶对基因组具有潜在危险,所以它们必须只在适当的受损部位发挥作用。

Double-Strand Breaks Are Efficiently Repaired

An especially dangerous type of DNA damage occurs when both strands of the double helix are broken, leaving no intact template strand to enable accurate repair. Ionizing radiation, replication errors, oxidizing agents, and other
双螺旋的两条链都断裂时,会发生一种特别危险的 DNA 损伤,没有完整的模板链来进行准确修复。电离辐射、复制错误、氧化剂和其他因素。
removal of covalent modifications from clamp, reloading of replicative DNA polymerase, continuation of accurate DNA synthesis metabolites produced in the cell cause breaks of this type. If these lesions were left unrepaired, they would quickly lead to the breakdown of chromosomes into smaller fragments and to loss of genes when the cell divides. However, two distinct mechanisms have evolved to deal with this type of damage by restoring an intact double helix: nonhomologous end joining and homologous recombination (Figure 5-45).
去除夹具上的共价修饰,重新装载复制 DNA 聚合酶,继续准确的 DNA 合成代谢产物在细胞中引起这种类型的断裂。如果这些损伤未修复,它们将迅速导致染色体分解为较小的片段,并在细胞分裂时导致基因丢失。然而,已经演化出两种不同的机制来处理这种类型的损伤,通过恢复完整的双螺旋:非同源末端连接和同源重组(图 5-45)。
The simplest to understand is nonhomologous end joining, in which the broken ends are processed to remove any damaged nucleotides and simply brought together and rejoined by DNA ligation, generally with the loss of nucleotides at the site of joining (Figure 5-46). This end-joining mechanism, which can be seen as a "quick and dirty" solution to the repair of double-strand breaks, is the predominant way of repairing these lesions in mammalian somatic cells. Although a change in the DNA sequence (a mutation) usually results at the site of breakage, so little of the mammalian genome is essential for life that this mechanism is apparently an acceptable solution to the problem of rejoining broken chromosomes. By the time a human reaches the age of 70, the typical somatic cell contains more than 2000 such "scars," distributed throughout its genome, representing places where DNA has been inaccurately repaired by nonhomologous end joining.
最简单理解的是非同源末端连接,其中断裂的末端被处理以去除任何受损核苷酸,然后简单地将它们放在一起,并通过 DNA 连接重新连接,通常在连接点丢失核苷酸(图 5-46)。这种末端连接机制可以被视为修复双链断裂的“快速而粗糙”的解决方案,是哺乳动物体细胞中修复这些损伤的主要方式。尽管在断裂点通常会导致 DNA 序列的变化(突变),但哺乳动物基因组中对生命至关重要的部分很少,因此这种机制显然是重新连接断裂染色体问题的可接受解决方案。当人类达到 70 岁时,典型的体细胞中包含超过 2000 个这样的“疤痕”,分布在其基因组中,代表 DNA 被非同源末端连接不准确修复的地方。
But nonhomologous end joining presents another danger: nonhomologous end joining can occasionally generate rearrangements in which one broken chromosome becomes covalently attached to another. This can result

Figure 5-44 How translesion DNA polymerases are recruited to damaged templates. According to this model, a replicative polymerase stalled at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated - see Figure 3-65), which releases the replicative DNA polymerase and, together with the damaged DNA, attracts a translesion polymerase specific to that type of damage. Once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the highfidelity replicative polymerase is brought back into play.
图 5-44 转录延伸 DNA 聚合酶如何被招募到受损模板。根据这个模型,停滞在 DNA 损伤点的复制聚合酶被细胞识别为需要救援。专门的酶会共价修饰滑动夹具(通常是泛素化的 - 见图 3-65),这会释放复制 DNA 聚合酶,并与受损 DNA 一起吸引特定于该类型损伤的转录延伸聚合酶。一旦受损 DNA 被绕过,夹具的共价修饰被去除,转录延伸聚合酶解离,高保真度的复制聚合酶再次发挥作用。
Figure 5-45 Cells can repair doublestrand breaks in one of two ways. (A) In nonhomologous end joining, the break is first "cleaned" by a nuclease that chews back the broken ends to produce flush ends. The flush ends are then stitched together by a DNA ligase. Some nucleotides are usually lost in the repair process, as indicated by the black lines in the repaired DNA. (B) If a double-strand break occurs in one of two duplicated DNA double helices after DNA replication has occurred, but before the chromosome copies have been separated, the undamaged double helix can be used as a template to repair the damaged double helix through homologous recombination. Although more complicated than nonhomologous end joining, this process accurately restores the original
图 5-45 细胞可以通过两种方式修复双链断裂。(A)在非同源末端连接中,断裂首先被核酸酶“清理”,核酸酶会咬掉断裂的末端以产生平滑末端。然后,DNA 连接酶将平滑末端缝合在一起。通常在修复过程中会丢失一些核苷酸,如修复后 DNA 中的黑线所示。(B)如果在 DNA 复制发生后,但染色体复制尚未分离之前,两个复制的 DNA 双螺旋中的一个发生双链断裂,那么未受损的双螺旋可以用作模板,通过同源重组修复受损的双螺旋。虽然比非同源末端连接更复杂,但这个过程可以准确恢复原始的。
in chromosomes with two centromeres and chromosomes lacking centromeres altogether; both types of aberrant chromosomes are missegregated during cell division. As previously discussed, the specialized structure of telomeres prevents the natural ends of chromosomes from being mistaken for broken DNA and "repaired" in this way.
在具有两个着丝点和完全缺乏着丝点的染色体中;这两种异常染色体在细胞分裂过程中都会被错误分离。正如先前讨论的那样,端粒的特殊结构防止染色体的自然末端被误认为是断裂的 DNA,并以这种方式“修复”。
A much more accurate type of double-strand break repair is also possible (see Figure 5-45B). Here, a damaged DNA molecule is repaired using a second DNA sequence at the site of the break. DNA double helix as a template, one with an identical (or nearly identical) DNA Homologous recombination is described in detail in the next part of this chapter. Although nonhomologous end joining and homologous recombination are the two principal ways that cells repair double-strand breaks, additional mechanisms exist.
双链断裂修复的一种更准确类型也是可能的(见图 5-45B)。在这种情况下,受损的 DNA 分子使用断裂点处的第二个 DNA 序列进行修复。DNA 双螺旋作为模板,其中一个具有相同(或几乎相同)的 DNA 同源重组在本章的下一部分中有详细描述。尽管非同源末端连接和同源重组是细胞修复双链断裂的两种主要方式,但还存在其他机制。
Figure 5-46 Nonhomologous end joining. (A) A central role is played by the Ku protein, a heterodimer that quickly grasps the broken chromosome ends. The additional proteins (shown in blue) are recruited to hold the broken ends together and remove any damaged nucleotides before the two DNA molecules are joined covalently by a specialized ligase that is dedicated to nonhomologous end joining. During this process, any single-strand gaps that arise are "filled in" by specialized repair polymerases. When DNA suffers double-strand breaks through ionizing radiation or chemical attack, the broken ends are often chemically damaged. Nonhomologous end joining is unusually versatile in being able to "clean up" just about any type of damaged end. (B) Three-dimensional structure of a Ku heterodimer bound to the end of a duplex DNA fragment. This Ku protein is also essential for joining, a specific process through which antibody and T-cell receptor diversity is generated in developing B and T cells (discussed in Chapter 24). V(D)J joining and nonhomologous end joining share many mechanistic similarities, but the former relies on specific double-strand breaks that are produced deliberately by the cell. (From J. Walker, R. Corpina, and J. Goldberg, Nature 412:607-614, 2001. With permission from Springer Nature; PDB codes: .)
图 5-46 非同源末端连接。(A) Ku 蛋白起着核心作用,它是一个迅速抓住断裂染色体末端的异源二聚体。其他蛋白质(蓝色显示)被招募来将断裂末端固定在一起,并在两个 DNA 分子被非同源末端连接的专门连接酶共价连接之前去除任何受损核苷酸。在这个过程中,任何产生的单链间隙都会被专门的修复聚合酶“填补”。当 DNA 遭受电离辐射或化学攻击导致双链断裂时,断裂末端通常会受到化学损伤。非同源末端连接在能够“清理”几乎任何类型的受损末端方面异常灵活。(B) 一个 Ku 异源二聚体与双链 DNA 片段末端结合的三维结构。这种 Ku 蛋白对