分子生物学教学课件：9 章 The Mutability and Repair of DNA

1、回顾第回顾第8章的内容章的内容Chapter 9 The Mutability and Repair of DNAChapter 92) Chapter 9Chapter 9Chapter 9Chapter 9OutlineChapter 9Chapter 9Chapter 9Chapter 91.How the replication errors are resulted?2.What is the nature of the replication errors? 3.How they are recognized and correctly repair?Chapter 9Chapte

2、r 9Figure 9-2 A mutation may be introduced by misincorporation of a base in the first round of replication. In the second round of replication, the mutation becomes permanently incorporated in the DNA sequence.Chapter 9Chapter 9Chapter 9Chapter 9Chapter 9Chapter 9Chapter 9Chapter 9These mutations mi

3、ght be caused by insertion of transposon (转座子转座子)or by aberrant action of cellular recombination processes. Rate of spontaneous mutation at any given site on chromosomal ranges from 10-6 to 10-11 per round of DNA replication, with some sites being “hotspot” . Chapter 9Chapter 9Figure 9-1 Base-change

4、 substitutionsChapter 9Chapter 9transitiontransversionmutation by base substration.swfATG,GTG,GAG,CCG,GCC,GAG,TAGATG,GTG,GAG,CTG,GCC,GAG,TAGATG,GTG,GAG,CCG,GCC,GAG,TAGATG,GTG,GAG,CG,GCC,GAG,TAGATG,GTG,GAG,CGG,CCG,AGT,AGPoint mutationInsertions or deletionsChapter 9Chapter 9addition and deletion muta

5、tion Chapter 9Chapter 9Fuctions of MutS:1. In E.Coli，MutS scans the DNA, recognizes the mismatch from the distortion they cause in the DNA backbone. Chapter 9Chapter 9Functions of MutSChapter 9Chapter 9Chapter 9Chapter 9Figure 9-4 MutS is a dimer. One monomer interacts with the mismatch specifically

6、, and the other nonspecifically. DNA is kinkedBaseBackboneATPChapter 9Chapter 9Further steps of mismatch repair, we must pay attention to the other two important parts of the mismatch repair system-MutL and MutH.Chapter 9Chapter 9Chapter 9Chapter 9(Dam) methylationChapter 9Chapter 9Chapter 9Chapter

7、9a. Replication generates hemimethylated DNA in E.coli.b. MutH makes incision in unmethylated daughter strand.Figure 9-5Chapter 9Chapter 9Figure 9-6Chapter 9Chapter 9mismatch_repair.swfChapter 9Chapter 9(自发的自发的)(水解水解)(转氨转氨)(烷基化烷基化)(氧化氧化)(辐射辐射)(嵌入嵌入)Chapter 9Chapter 9Figure 9-7Deamination CUHydrolysi

8、s creates apurinic deoxyribose Deamination 5-mC T Chapter 9Chapter 9Can 5-mC T lesion be repaired?Why DNA contains T instead of U?Chapter 9Chapter 9DNA is subject to attack from Reactive oxygen species (O2-, H2O2, OH)Figure 9-8 G modificationAlkylating chemical: Nitrosamines (亚硝胺亚硝胺)“O2-” hyperoxide

9、“H2O2” Peroxide“OH” hydroxylChapter 9Chapter 9Figure 9-9 Thymine dimer.相邻两个嘧啶之间发生光化学聚合相邻两个嘧啶之间发生光化学聚合, 形成胸腺嘧啶二聚体形成胸腺嘧啶二聚体(环丁烷环环丁烷环)。 Chapter 9Chapter 93. Base analogs (碱基类似物碱基类似物) and intercalating agents (嵌入剂嵌入剂)(5-BrU) (溴尿嘧啶溴尿嘧啶)Chapter 9Chapter 9Chapter 9Chapter 9Figure 9-10a Base analogues烯醇异构体酮

10、异构体A:T C:GChapter 9Chapter 9Intercalating agents are flat molecules containing several polycyclic rings that interact with the normal bases in DNA through hydrogen bonds and base stacking.溴乙非啶二氨基吖啶/原黄素吖啶, 氮蒽Figure 9-10b Base analoguesChapter 9Chapter 9Chapter 9Chapter 91.Direct reversal of DNA damag

11、e by photoreactivation (光激活作用光激活作用) and alkyltransferase (烷基转移酶烷基转移酶)2.Base excision repair (碱基切除修复碱基切除修复)3.Nucleotide excision repair4.Recombination (DSB) repairs5.Direct joining repairs6.Translesion DNA synthesisChapter 9Chapter 9Figure 9-11 PhotoreactivationMonomerization of thymine dimers by DNA

12、 photolyases in the presence of visible light.DNA光解酶光解酶 Chapter 9Chapter 9Figure 9-12 Methyl group removal.Methyltransferase catalyzes the transfer of the methyl group on O6-methylguanine to cystein residue (半胱氨酸半胱氨酸)on the enzyme, thereby restoring the normal G in DNA.direct_repair.swfGlycosylase (

13、糖基化酶糖基化酶) Chapter 9Chapter 9The damaged base which is flipped out The enzymeThe DNAFigure 9-14: base-flipping recognition by glycosylaseChapter 9Chapter 9Chapter 9Chapter 9细胞中有多种具有不同特异性的细胞中有多种具有不同特异性的DNA糖基化酶。分别糖基化酶。分别可以识别并去除损伤碱基。目前已经知道有可以识别并去除损伤碱基。目前已经知道有8种不同种不同的的DNA糖基化酶糖基化酶。 1. The AP site is creat

14、ed by the hydrolysis of glycosylase bond.2. AP endonuclease & exonuclease cut out the 5 phosphate.3. DNA polymerase fill in the gap. Chapter 9Chapter 9Figure 9-13Chapter 9Chapter 9oxoG:A repair. A glycosylase recognizes the mispair and removes A. A fail-safe glycosylase also removes T from T:G mispa

15、irs, as if it knows how T is produced. Figure 9-15:防错糖基化酶防错糖基化酶 给给DNA糖基化酶糖基化酶除去修饰的碱基提除去修饰的碱基提供了第二次机会供了第二次机会 base excision repair.swfChapter 9Chapter 9What is the difference between the two kinds of excision repair systems?How does the nucleotide work?Chapter 9Chapter 91.Recognize distortions to the

16、shape of the DNA double helix2.Remove a short single-stranded segment that includes the lesion. 3.DNA polymerase/ligase fill in the gap.与碱基切除修复不同，核苷酸切除修复酶不能区分各种不同的与碱基切除修复不同，核苷酸切除修复酶不能区分各种不同的损伤，而是识别双螺旋形状上的扭曲。损伤，而是识别双螺旋形状上的扭曲。 Chapter 9Chapter 91) Once encountering a distortion UvrA exits the complex

17、and UvrB melts the DNA to create a single-strand bubble around the lesion.2) Next, UvrB recruits UvrC, and UvrC creates two incisions in different positions on one strand.3) Finally, DNA polymerase and ligase fill in the gap.Figure 9-16*nucleotide excision repair.swfChapter 9Chapter 9Nucleotide exci

18、sion repair (NER) system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template Figure 9-17. Transcription-couple repair.核苷酸切除修复不仅能修复整个基因核苷酸切除修复不仅能修复整个基因组中的损伤，而且当组中的损伤，而且当RNA聚合酶的转聚合酶的转录过程被某个基因发生转录链损伤终录过程被某个基因发生转录链损伤终止时，可进行转录偶联修复止时，可进行转录偶联修复（transc

19、ription-coupled repair）。）。此种修复主要是将核苷酸切除修复蛋此种修复主要是将核苷酸切除修复蛋白募集到受阻的白募集到受阻的RNA聚合酶上。聚合酶上。 Chapter 9Chapter 9Double-strand break (DSB) repair pathwayDetails are in chapter 10Chapter 9Chapter 95. Direct joining repairs NHEJ: Nonhomlogous end joining V(D)JChapter 9Chapter 9 Error- prone repair* Occurs when

20、 the above repairs are not efficient enough so that a replicating polymerase encounters a lesion Translesion synthesis is also called a fail-safe or last resort mechanism. Chapter 9Chapter 9 Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly acr

21、oss the damage site. Translesion polymerase is produced by cell in response to the DNA damage Translesion polymerases are expressed as part of the SOS response pathway. Chapter 9Chapter 9Crystal structure of a translesion polymerase.FIGURE 9-20 A Y-family polymerase found in many organisms. Chapter

22、9Chapter 9Translesion DNA synthesis in E. coliFIGURE 9-19 Translesion DNA synthesis in E. coli 复制时一旦遇到损伤，复制时一旦遇到损伤，DNA聚合酶聚合酶III与其滑动夹与其滑动夹一起从一起从DNA上脱落，并被上脱落，并被移损移损DNA聚合酶所取代。聚合酶所取代。移损移损DNA聚合酶越过受损聚合酶越过受损部位（胸腺嘧啶二聚体）部位（胸腺嘧啶二聚体）进行进行DNA合成，然后移损合成，然后移损聚合酶被聚合酶被DNA聚合酶聚合酶III替替换。换。 DNA修复（修复（DNA repairing）是细胞对）是细

23、胞对DNA受损伤后的一受损伤后的一种反应，这种反应可能使种反应，这种反应可能使DNA结构恢复原样，重新能执行结构恢复原样，重新能执行它原来的功能；但有时并非能完全消除它原来的功能；但有时并非能完全消除DNA的损伤，只是的损伤，只是使细胞能够耐受着使细胞能够耐受着DNA的损伤而能继续生存。的损伤而能继续生存。虽然直接修复以及切除修复能够完全消除虽然直接修复以及切除修复能够完全消除DNA的损伤，而的损伤，而在实际中，在实际中，DNA经常发生双链断裂。如果对这种断裂不能经常发生双链断裂。如果对这种断裂不能进行及时的修复，就会使细胞产生致命性的后果。对于大进行及时的修复，就会使细胞产生致命性的后果。对

24、于大多数细胞而言，用于修复双链断裂的主要机制就是同源重多数细胞而言，用于修复双链断裂的主要机制就是同源重组（组（Homologous Recombination)。部分可以通过非。部分可以通过非同源末端连接（同源末端连接（NHEJ）的方式进行修复。）的方式进行修复。 Chapter 9Chapter 9Summary and key points All the repair mechanisms (details) and the cause of the corresponding DNA errors and DNA damagesMismatch repair system: DNA

25、replication errorsDirect reversal of DNA damages: utraviolet (UV) irradiation induced thymine/pyrimidine dimmers-photoactivation; alkylation agents caused O6-methylguanine-methyl transferase.Base excision repair: the base damage by alkylation and oxidation Nucleotide excision repair: the distortion

26、of the DNA double helix by thymine dimmer or the bulky chemical adduct (加合物) on a base.Recombination repair: double-strand breaks in DNA, errors encountered by a replication fork.Translesion synthesis allows the replication to proceed across DNA damage at a cost of error-prone replication. A different D