真核生物DNA修复机制 董霞 蒋舜媛 程在全.

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1 真核生物DNA修复机制 董霞 蒋舜媛 程在全

2 错配 复制过程 紫外线 化学试剂 电离辐射 DNA损伤 进化 致病、致死 修复

3 真核生物DNA的修复机制 重组修复 非同源末端连接修复 跨越修复 切除修复

4 一. 真核生物DNA损伤的重组修复 同源重组的重要性 染色体重组及重组修复的有关蛋白 酵母影响同源间重组的途径
酵母菌中复制导致的染色体片段化 酵母菌重组修复的机制 脊椎动物细胞的重组修复

5 同源重组的重要性 According to the participants of the National Academy of Sciences Colloquium entitled "Links Between Recombination and Replication: Vital Roles of Recombination," organized by Charles Radding (chair), Nicholas Cozzarelli, Michael Cox, Kenneth Marians, and James Haber and held at the Beckman Center of the Academy in Irvine, California, on November 10-12, 2000.

6  The recent surge in works on interdependence of DNA replication and homologous recombination, conducted in experimental systems ranging from bacteriophages to mammalian cell lines, highlighted faltering replication forks as the connecting points between the two seemingly opposite domains of DNA metabolism.

7 Proc. Natl. Acad. Sci. USA, Vol
Proc. Natl. Acad. Sci. USA, Vol. 98, Issue 15, , July 17, Colloquium Paper DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination Andrei Kuzminov*

8 染色体重组及重组修复的有关蛋白 染色体的同源重组是重组修复的基础。(fig32) 酵母中重组相关蛋白与phage T4、大肠杆菌的比较，通过对重组缺陷突变体的研究,揭示DNA代谢在跨不同领域的明显的相似性，分析了相应重组酶及有关蛋白质的特征(fig33) 如PCNA(fig38)。当单链DNA断裂不能复制时，就引发同源重组。

9 酵母中影响同源性重组的途径 A. RAD51/54/55/57 作用于同源重组之间,强烈影响同源染色体间的重组.
B. rad50, mre11 (rad58), rad59, and xrs2 (rad60),作用于姊妹重组之间, RAD50/58/59/60途径轻微提高同源间重组的水平. 它们都受RAD52的控制

10 酵母菌中复制导致的 染色体片段化 A:点检测 真核生物复制及修复过程中精确的点检测方式阻止了细胞周期以避免DNA的明显损伤，阻止停滞不前的复制叉的大量出现，从而产生染色体片段化，然后或细胞停止分裂或有助于重组修复的形成。

11 Mec1是一个依赖于RAD52 的 DNA损伤的点检测关键蛋白 .它 的突变在新合成DNA中积累单链断裂

12 B: 当出芽酵母细胞用羟基尿抑制核苷酸还原酶, 细胞中的DNA会集聚单链断裂
B: 当出芽酵母细胞用羟基尿抑制核苷酸还原酶, 细胞中的DNA会集聚单链断裂.单链断裂转换为双链断裂也使DNA片段化，这一些现象最直接的解释是原来有单链断裂的DNA上复制叉解散(Fig. 2 B-- C), 出现断裂双链。

13 rad27 pol30 rfc1 cdc9 C:冈崎片段不能成熟的突变导致DNA片段化 DNA单链断裂积累 冈崎片段不能成熟突变
致 死

14 D:Michael Resnick 和Dmitry Gordenin 报道了一系列DNA聚合酶δ突变体中，如果再加上rad27突变，缺乏3’—5’核酸外切酶活性,这种突变是致死的 。但是, DNA聚合酶δ突变体中,如果具有Rad27和 3’ - 5’ Exo of Pol（聚合酶的3’ - 5’核酸外切酶活性）,这样的突变体仍能存活，不过会发生超重组，超重组依赖于RAD50, RAD51, 和 RAD52. 这种情况下冈崎片段有可能不能成熟, 也出现DNA片段化。

15 酵母菌重组修复的机制 A、暂停复制叉修复的假说(fig)

16 B、链退火修复复制叉 依靠单链退火修复复制叉的两种途径 复制叉到达模板DNA的单链断裂后解体, Rad52促进染色质上带有单链裂缝的分离末端的重新退火。 单链断裂的修复 全长染色质上的单链断裂得到填补，Rad54催化完整染色质在双链末端临近位置的解链。 Rad52催化一条单链与打开的姊妹链的双链之一的末端退火，从而产生复制叉结构。(fig35)

17 C:酵母菌的双链断裂重组 酵母菌的双链断裂重组有两个阶段：1、末端之一侵入到同源染色体，形成类似于复制叉的重组中间体，2、另一末端的聚集阻止了重组中间体形成复制叉. 如果因为同源性有限，第二个末端不能与重组中间体结合，复制叉成熟，继而推进到染色体末端。

18 D、双链末端修复的发生顺序假说 双链末端被Rad50/Rad58/Rad60加工产生ssDNA突出，与RPA形成复合体;
Rad52 催化RPA 与Rad51的转化； Rad51, 在 Rad54, Rad55, 和Rad57的帮助下，催化双链末端侵入完整的姊妹双链。 在这一机制中, Rad51作为蛋白质复合体的组成者和催化剂两方面起着重要的作用(fig41)。

19 脊椎动物细胞的重组修复 重组修复机制与酵母大致相同，只是要复杂些。多数双链断裂被同源重组如实修复，这一重组机制是通过缩短断裂一端的同源 序列长度 实验证实的：得到的修复产物携带着断裂的非同源一端的多种拷贝。在复制区内及前后，有很多微小同源区。显然，断裂的同源末端侵袭完整的姊妹染色单体，启动复制叉，产生的末端在微小同源区与非同源末端相连。

20 Proc. Natl. Acad. Sci. USA, Vol
Proc. Natl. Acad. Sci. USA, Vol. 98, Issue 15, , July 17, Colloquium Paper Homologous DNA recombination in vertebrate cells Eiichiro Sonoda*, , Minoru Takata*, , Yukiko M.

21 其它差异： A、用鸡淋巴瘤细胞株为材料做的突变实验，发现不同于酵母的两个主要变异：1、rad51突变脊椎动物细胞迅速死亡，死之前发育中的全部染色体发生变异。2、脊椎动物的rad52突变体细胞对DNA损伤剂不是太敏感。 B、脊椎动物与酵母的另一个差异是：脊椎动物中存在多种共生同源的重组酶蛋白Rad51p。XRCC2是Rad51p的一个共生同源物，但非同源末端的连接不受影响。XRCC3是 Rad51p的另一个共生同源物，通过同源重组对双链突变的修复需要它。

22 同 源 重 组 DNA复制使先前存在的 DNA损伤恶化，因而阻碍了以后 DNA的复制。需要同源重组帮助修复
同 源 重 组 DNA复制使先前存在的 DNA损伤恶化，因而阻碍了以后 DNA的复制。需要同源重组帮助修复 DNA损伤引起复制叉失控使复制体具有断裂的倾向 同源重组使单链断裂和双链断裂经过复制叉的解体和重新形成而得到修复。 真核生物重组酶对真核生物单链退火有较大的作用。 近期的报道认为,RAD51在真核中起关键作用.

23 二. 非同源末端连接(NHEJ)修复 A2 B2 同源片段A1 /A2 ; 同源片段B1/B2
如果A1的末端和B1的末端连接,可进行非同源末端连接修复,依靠Ku类蛋白质,而不是象HR修复依靠RAD52非等位基因编码的蛋白质. 事实上,在NHEJ中,仍然存在着一些微小的同源小区域, 帮助NHEJ. 在酵母菌和脊椎动物中, DSB修复途径存在一些差异.

24 HR、NHEJ在酵母菌和脊椎动物中DSB的作用 NHEJ HR 酵母菌 + +++ 脊椎动物 +++ +

25 三. 跨越修复 跨越修复 在RPA、Rad、 DNA聚合酶、 PCNA等的作用下，使新生的DNA跨越模 板上错误的碱基而继续合成。
三. 跨越修复 跨越修复 在RPA、Rad、 DNA聚合酶、 PCNA等的作用下，使新生的DNA跨越模 板上错误的碱基而继续合成。 最近新发现的UmuC-PinB-Rad30-Rev1 复合体在三种生物域都存在. 它们在跨越损伤修复中起重要作用. 机制一(fig36) 机制二(fig39)

26 Biochem.Soc Trans May29(Pt 2):187-91
Eukaryotic mutagenesis and translesion replication dependant on DNA polymerase zeta and Rev1 protein

27 四. 切除修复 切除修复是一种多步骤的酶反应过程(fig37) 核酸内切酶将损伤的DNA切除 由DNA聚合酶进行聚合反应填补修复的切口
切除修复分为核苷酸切除修复(NER)和碱基切除修复(BER). 而BER又分为短补丁(1个核苷酸)修复和长补丁(2-10个核苷酸)修复.

28 Mutut Res 2001.Sep.4.486(4):217-47 Repair of DNA interstrand cross-links DNA interstrand interstrand cross-links (TCLs) are very toxic to dividing cells 多种途径如NER、HR转录后复制/跨损伤修复都参与DNA的ICLs修复

29 Crit Rev Biochem md. Biol.2001 36(4):337-97
Molecular mechanisms of DNA damage and repair: progress in plants 植物主要受OV-B射线影响，导致过氧化损伤和交叉连接（DNA-Pr、DNA-DNA）修复方式有：直接反转、BER IVER 光修复、跨越修复、双链断裂途径错配修复途径 直接反转修复、光修复只需要1种蛋白参与，其它修复方式要求多种蛋白参与。 双链断裂需要HRR或NHEJ，而在植物中，NHEJ导致错误修复几率比其它真核生物的高，从而产生进化。BER：单核苷切除途径：依赖DNA polß,多核苷（2-10nt）切除：需PCNA

30 PCNA: proliferating cell nuclear antigen : ringmaster of the genuine
Cell Death Differ 2001.Nov.8(11): Functional interactions and signaling properties of mammalian DNA mismatch repair proteins PCNA: proliferating cell nuclear antigen : ringmaster of the genuine PCNA是决定细胞存活与死亡的关键分子之一，PCNA基因可由P53诱导。如果PCNA量丰富，无P53，进行DNA复制；如果PCNA量丰富，有P53，进行DNA修复；如果PCNA量低，无P53，细胞编程序性死亡。PCNA：低等原核生物中只是滑动夹状蛋白，而在真核生物中成为决定细胞命运的关键分子。(fig38)

31 Mutut Res 2001.Sep.4.486(4):217-47 Repair of DNA interstrand cross-links DNA interstrand interstrand cross-links (TCLs) are very toxic to dividing cells 多种途径如NER、HR转录后复制/跨损伤修复都参与DNA的ICLs修复

32 Fig. 1. DNA replication vs. homologous recombination
Fig. 1.   DNA replication vs. homologous recombination. Chromosomes are shown as double lines. Parental strands are filled; daughter strands are open. (A) A chromosome. (B) Chromosome replication has been initiated. (C) Chromosome replication is nearing completion. (D) Chromosome replication is complete. (E) Strand degradation in preparation for homologous recombination has started. (F) Strand degradation is nearing completion, whereas annealing of the complementary strands is going on.

33 Activity T4 E.coli Budding yeast
Replicative DNA polymerase gp43 DNA polIII DNA pol Sliding clamp gp45 DnaN(ß) PCNA Clamp loader gp44/46 Complex RFC Replicative helicase gp41 DnaB MCM Replicative primase gp61 DnaG ss-DNA-binding protein gp32 SSB RPA Producing 3’-ssDNA tails gp46/47 RecBCD Rad50/58/60 Anti-SSB activity UvsY RecBCD or Rad52/59 RecFOR Recombinase UvsX RecA Rad51 Recombinase regulators ? Rad55/57 Auxiliary helicases UvsW RecG Rad54/Tid1 DNA junction resolution gp49 RuvABC fig

34 Fig. 2.   The pathways of replication fork stalling/disintegration with subsequent resetting/repair. DNA duplexes are shown as double lines; a protein tightly bound to DNA is shown as a bricked circle. For all Holliday junctions, one of the two possible resolution directions is indicated by the small arrows. (A) A replication fork. (B) The replication fork approaching a single-strand interruption in template DNA. (C) The replication fork has collapsed at the interruption. (D) Double-strand end invasion to restore the replication fork structure. (E) A stalled replication fork. (F) Regression of the stalled replication fork forms a double-strand end and a Holliday junction. (G) Double-strand end invasion to restore the replication fork structure. Resolution of the Holliday junction in F leads to replication fork breakage (C). Resolution of the Holliday junctions in D or G, or exonucleolytic degradation of the linear tail in F leads to restoration of the replication fork structure (A). fig34 Fig 34a

35 Fig. 3.   Two ways to repair a replication fork by single-strand annealing. DNA duplexes are shown as double lines; sister chromatid cohesion is indicated by thin dumbbells. (A) A replication fork approaching a single-strand interruption in template DNA. (B) The replication fork has collapsed. (C) Rad52-promoted reannealing of the detached end with the complementary single-strand gap on the full-length chromatid. (D) Repair of the single-strand interruption. (E) Filling-in the single-strand gap on the full-length chromatid. Sister chromatid alignment is shown. The thin arrows indicate a hypothetical signal from the Rad52-bound double-strand end to the intact sister chromatid. (F) Rad54-catalyzed unwinding of the intact chromatid in the vicinity of the double-strand end. (G) Rad52-catalyzed annealing of the double-strand end with the open sister duplex to generate a replication fork structure.

36 Fig. 1. Model of SOS translesion replication by DNA polymerase V
Fig. 1.   Model of SOS translesion replication by DNA polymerase V. The two DNA strands are shown as green lines, and the replication-blocking lesion is represented by the red rectangle. The three major steps in TLR are pre-initiation (2), in which the RecA nucleoprotein filaments assembles; initiation (3 and 4), which involves binding of pol V to the primer-template and loading of the  subunit clamp; and lesion bypass by pol V holoenzyme (5). SSB is suggested to help in displacing RecA from DNA both at the initiation and lesion bypass steps.

37 Figure 1. Model for the sequential assembly of the various components of nucleotide excision repair. Global genome repair involves the initial binding of the XPC-hHR23B and XPE binding proteins followed by downloading of the TFIIH transcription and helicase complex that remodels the damaged site. Transcription coupled repair involves initial response to damage by stalling of the RNA polymerase II apparatus, and coupling by the CSA and CSB proteins. Subsequent steps proceed in common, consisting of loading the XPG nuclease, the XPA-RPA DNA binding proteins, and the ERCC1-XPF nuclease. After nuclease cleavage around the dimer site, the excision complex departs and the site is resynthesized by PCNA-polymerase delta and ligase I. Figure has been redrawn (37) with addition of the transcription repair pathway.

38 Fig. 1. DNA sliding clamps from E. coli, human, and phage T4
Fig. 1.   DNA sliding clamps from E. coli, human, and phage T4. Ribbon diagrams are shown for sliding clamps from different organisms: (A) E. coli  (4), (B) human PCNA (6), and (C) phage T4 gp45 (7). Each monomeric unit of the ring is shown in a different color. The arrows indicate the protomer interfaces. (D) The scheme illustrates assembly of a  ring on a primed template by  complex in an ATP-driven reaction, followed by association of the core polymerase with  for processive DNA synthesis.

39 Fig. 4. RFR in UV-irradiated cells (adapted from refs. 76, 80, and 92)
Fig. 4.   RFR in UV-irradiated cells (adapted from refs. 76, 80, and 92). The replication fork is blocked by a UV photo-product (black triangle) in the leading-strand template. RFR, proposed to be catalyzed by RecG in E. coli (76), or by Rad51 (the yeast RecA homologue) in S. pombe (80), renders the damaged DNA double stranded and thereby allows direct repair by nucleotide excision repair enzymes (A). If the lagging-strand polymerase has continued synthesis past the lesion, leading-strand DNA synthesis using the lagging strand as template followed by reverse branch migration [proposed to be catalyzed by RecG in E. coli (76) and by Rqh in S. pombe (80)] reconstitutes a fork on which the lesion has been bypassed (B; ref. 94). Full and dashed lines represent the template and the newly synthesized DNA, respectively; the arrowhead indicates the 3' end of the growing strand.

40 RPA Rad51 Fig. 1.   Generic model for assembly of recombinase on ssb-coated ssDNA. (A) Tracts of ssDNA form because of resection at DSB sites or stalling of polymerase. (B) ssb assembles into oligomeric filaments on tracts of ssDNA, removing secondary structures. (C) Mediator protein binds to ssb-coated DNA, causing a local remodeling of the ssb filament. (D) Recombinase initiates filament formation at sites of mediator-ssb-ssDNA. Recombinase filaments then elongate displacing ssb. (E) The elongated recombinase filament searches for homologous sequences. (F) Strand exchange occurs. The outgoing ssDNA strand is bound by ssb.

41 Fig. 4.   Model for mediator-dependent coupling of ssDNA strand resection and recombinase assembly at DSBs during meiosis. (A) Strand-specific nuclease loads at DSB sites. (B) Mediators are loaded on DNA ends before nuclease proceeds a distance far enough to allow cytologically detectable amounts of RPA. (C) Rad51 is recruited to ends via mediator interactions, again before nuclease has proceeded far from the end. (D) Nucleolytic resection of DNA occurs in concert with elongation of the Rad51 filament. (E) Rad51 carries out homology search. (F) Strand exchange occurs with RPA loading on the outgoing ssDNA strand.

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44 谢 谢!

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47 D:Dna2p 与 Rad27p形成复合体一起加工冈崎片段。dna2ts突变体在仅次于致死温度的情况下依赖于RAD52基因。在裂殖酵母中，dna2基因的失活会导致染色体片段化
E: cdc24突变体在有染色体片段化信号下完成染色体复制后，突变体停止生长，dna2+, cdc24可以通过pcn1+ (DNA clamp) and rfc1+ (DNA clamp loader)基因而得到挽救。在离体条件下Cdc24p 结合到Pcn1p and Rfc1p上。冈崎片段不能成熟有可能导致cdc24突变型的多型化

48 酵母菌重组的酶学 Rad51 催化单链侵入 Rad52 促进退火 Rad53 Rad54 Rad55 Rad51 Rad57 Rad51