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

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 9Chapter 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,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 some sites being“hotspot”.DNA microsatellites(微卫星微卫星DNA)：These sequences(1)are important in human genetics and disease；(2)hard to be copied accurately and highly polymorphic(多态性多态性)in the population.Chapter 9Chapter 9Chapter 9Chapter 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 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,and the other nonspecifically.DNA is kinkedBaseBackboneATPChapter 9Chapter 9Further steps of miamatch 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 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 CUHydrolysis 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“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,形成胸腺嘧啶二聚体(环丁烷环)。Chapter 9Chapter 9Base 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烯醇异构体酮异构体Chapter 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 9Direct reversal of DNA damage by photoreactivation(光活化作用光活化作用)and alkyltransferase(烷基转移酶烷基转移酶)Base excision repair(切割修复切割修复)Nucleotide excision repairRecombination(DSB)repairsDirect joining repairsTranslesion 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 cystein 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细胞中有多种具有不同特异性的DNA糖基化酶。分别可以识别并去除损伤碱基。目前已经知道有8种不同的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 fail-safe glycosylase also removes T from T:G mispairs,as if it knows how T is produced.Figure 9-15:防错糖基化酶 给DNA糖基化酶除去修饰的碱基提供了第二次机会 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 the 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 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 excision 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（transcription-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 9Chapter 9Chapter 9 Error-prone repair*Occurs when 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 across 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 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。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 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 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 DNA polymerase is utilized.