分子生物學(xué)教學(xué)課件：CHAPTER 9 The Mutability and Repair of DNA

1、Chapter 9 The Mutability and Repair of DNAChapter 9Chapter 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 9Chapter 9Cha

2、pter 9Chapter 9Figure 9-1 Base-change substitutionsChapter 9Chapter 9transitiontransversionmutation by base substration.swfChapter 9Chapter 9These mutations might be caused by insertion by transposon or by aberrant action of cellular recombination processes.ATG,GTG,GAG,CCG,GCC,GAG,TAGATG,GTG,GAG,CTG

3、,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 mutation Rate of spontaneous mutation at any given site on chromosomal ranges from 10-6 to 10-11 per round of DNA replication, with

4、some sites being “hotspot” . DNA microsatellites (微衛(wèi)星微衛(wèi)星DNA)：These sequences (1) are important in human genetics and disease； (2) hard to be copied accurately and highly polymorphic (多態(tài)性多態(tài)性) in the population.Chapter 9Chapter 9Chapter 9Chapter 9Figure 9-2 A mutation may be introduced by misincorpora

5、tion 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 9Fuctions of MutS:1. In E.Coli，MutS scans the DNA, recognizes the mismatch from the distortion they cause in the

6、DNA backbone. Chapter 9Chapter 9Functions of MutSChapter 9Chapter 9Chapter 9Chapter 9Figure 9-4 MutS is a dimer. One monomer interacts with the mismatch specifically, and the other nonspecifically. DNA is kinkedBaseBackboneATPChapter 9Chapter 9Further steps of miamatch repair, we must pay attention

7、to the other two important parts of the mismatch repair system-MutL and MutH.Chapter 9Chapter 9Chapter 9Chapter 9(Dam)methylationChapter 9Chapter 9Chapter 9Chapter 9a. Replication generates hemimethylated DNA in E.coli.b. MutH makes incision in unmethylated daughter strand.Figure 9-5Chapter 9Chapter

8、 9Figure 9-6Chapter 9Chapter 9mismatch_repair.swfChapter 9Chapter 9(自發(fā)的自發(fā)的)(水解水解)(轉(zhuǎn)氨轉(zhuǎn)氨)(烷基化烷基化)(氧化氧化)(輻射輻射)(嵌入嵌入)Chapter 9Chapter 9Figure 9-7Deamination CUHydrolysis creates apurinic deoxyribose Deamination 5-mC T Chapter 9Chapter 9Can 5-mC T lesion be repaired?Why DNA contains T instead of U?Chapte

9、r 9Chapter 9DNA is subject to attack from Reactive oxygen species (O2-, H2O2, OH)Figure 9-8 G modificationAlkylating chemical: Nitrosamines (亞硝胺亞硝胺)“O2-” hyperoxide“H2O2” Peroxide“OH” hydroxylChapter 9Chapter 9Figure 9-9 Thymine dimer.相鄰兩個(gè)嘧啶之間發(fā)生光化學(xué)聚合, 形成胸腺嘧啶二聚體(環(huán)丁烷環(huán))。 Chapter 9Chapter 9Base analogs

10、(堿基類似物堿基類似物) and intercalating agents (嵌入劑嵌入劑)(5-BrU) (溴尿嘧啶溴尿嘧啶)Chapter 9Chapter 9Chapter 9Chapter 9Figure 9-10a Base analogues烯醇異構(gòu)體酮異構(gòu)體Chapter 9Chapter 9Intercalating agents are flat molecules containing several polycyclic rings that interact with the normal bases in DNA through hydrogen bonds and

11、base stacking.溴乙非啶二氨基吖啶/原黃素吖啶, 氮蒽Figure 9-10b Base analoguesChapter 9Chapter 9Chapter 9Chapter 9Direct reversal of DNA damage by photoreactivation (光活化作用光活化作用) and alkyltransferase (烷基轉(zhuǎn)移酶烷基轉(zhuǎn)移酶)Base excision repair (切割修復(fù)切割修復(fù))Nucleotide excision repairRecombination (DSB) repairsDirect joining repairsT

12、ranslesion DNA synthesisChapter 9Chapter 9Figure 9-11 PhotoreactivationMonomerization of thymine dimers by DNA photolyases in the presence of visible light.DNA光解酶 Chapter 9Chapter 9Figure 9-12 Methyl group removal.Methyltransferase catalyzes the transfer of the methyl group on O6-methylguanine to cy

13、stein residue on the enzyme, thereby restoring the normal G in DNA.direct_repair.swfGlycosylase (糖基化酶糖基化酶) Chapter 9Chapter 9The damaged base which is filpped out The enzymeThe DNAFigure 9-14: base-flipping recognition by glycosylaseChapter 9Chapter 9Chapter 9Chapter 9細(xì)胞中有多種具有不同特異性的DNA糖基化酶。分別可以識(shí)別并去除

14、損傷堿基。目前已經(jīng)知道有8種不同的DNA糖基化酶。 1. The AP site is created 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

15、 fail-safe glycosylase also removes T from T:G mispairs, as if it knows how T is produced. Figure 9-15:防錯(cuò)糖基化酶 給DNA糖基化酶除去修飾的堿基提供了第二次機(jī)會(huì) Chapter 9Chapter 9What is the difference between the two kinds of excision repair systems?How does the nucleotide work?Chapter 9Chapter 91.Recognize distortions to th

16、e shape of the DNA double helix2.Remove a short single-stranded segment that includes the lesion. 3.DNA polymerase/ligase fill in the gap.與堿基切除修復(fù)不同，核苷酸切除修復(fù)酶不能區(qū)分各種不同的與堿基切除修復(fù)不同，核苷酸切除修復(fù)酶不能區(qū)分各種不同的損傷，而是識(shí)別雙螺旋形狀上的扭曲。損傷，而是識(shí)別雙螺旋形狀上的扭曲。 Chapter 9Chapter 91) Once encountering a distortion UvrA exits the comple

17、x 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 ex

18、cision 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.核苷酸切除修復(fù)不僅能修復(fù)整個(gè)基因核苷酸切除修復(fù)不僅能修復(fù)整個(gè)基因組中的損傷，而且當(dāng)組中的損傷，而且當(dāng)RNA聚合酶的轉(zhuǎn)聚合酶的轉(zhuǎn)錄過(guò)程被某個(gè)基因發(fā)生轉(zhuǎn)錄鏈損傷終錄過(guò)程被某個(gè)基因發(fā)生轉(zhuǎn)錄鏈損傷終止時(shí)，可進(jìn)行轉(zhuǎn)錄偶聯(lián)修復(fù)止時(shí)，可進(jìn)行轉(zhuǎn)錄偶聯(lián)修復(fù)（tran

19、scription-coupled repair）。）。此種修復(fù)主要是將核苷酸切除修復(fù)蛋此種修復(fù)主要是將核苷酸切除修復(fù)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 9Chapter 9Chapter 9 Error-prone repair* Occurs when the above repairs are not efficient enough so that a replica

20、ting 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 across the damage site. Translesion polymerase is produced by ce

21、ll 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 9Chapter 9Translesion DNA synthesis in E. coliFIGURE 9-19 Translesion DNA synthesis in E. coli 復(fù)制時(shí)一旦遇到損傷，復(fù)制時(shí)一旦遇到損傷，DNA聚合酶聚合酶III與其滑動(dòng)夾與其滑動(dòng)夾一起從一起從DNA上脫落，并被上脫落，并被移損移損DNA聚合酶所取代。聚合酶所取代。移損移損DNA聚合酶越過(guò)受損聚合酶越過(guò)受損部位（胸腺嘧啶二聚體）部位（胸腺嘧啶二聚體）進(jìn)行進(jìn)行DNA合成，然后移損合成，然后移損聚合酶被聚合酶被DNA聚合酶聚合酶III替替換。換。 Chapter 9Chapter 9Summary and key points All the repair mechanisms