chapter9ThemutabilityandrepairofDNA.ppt

1、Welcome to My Molecular Biology Class,Molecular Biology of the Gene, 5/E - Watson et al. (2004),Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods,3/22/05,Part II: Maintenance of the Genome,Dedicated to the structu

2、re of DNA and the processes that propagate, maintain and alter it from one cell generation to the next,Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molec

3、ular level Ch 11: Site-specific recombination and transposition of DNA,3/22/05,The consequence of high rates of mutation,Mutation in germ line (生殖细胞) would destroy the species Mutation in soma (体细胞) would destroy the individual.,Maintenance of the correctness of the DNA sequence is definitely crucia

4、l for living organisms. Keeping the error rate as low as 10-10 (page191) is very expensive.,Build up a serious attitude to science!,I absolutely do not agree with Waston et al. at the points described 3rd it is responsible for diversity inside of species. Transposition (转座) is different from mutatio

5、n and recombination because (1) producing mechanism is different; (2) no mechanism to correct it; (3) existing in nature in a well-controlled manner (10-5). Not repaired but controlled.,CHAPTER 9: The mutability and repair of DNA,Molecular Biology Course,Replication errors and their repair DNA damag

6、e Repair of DNA damage,3 teaching hours,Two important sources for mutation (unavoidable),Inaccuracy in DNA replication (10-7 is not accurate enough) Errors (错误) Chemical damage to the genetic material (environment) Lesions (损害,伤害arose from spontaneous damage) Damage (损害,伤害 caused by chemical agents

7、and radiation,To repair an error or damage,First, Detect the errors Second, Mend/repair the errors or lesions in a way to restore the original DNA sequence.,Questions to be addressed,How is the DNA mended rapidly enough to prevent errors from becoming set in the genetic material as mutation How does

8、 the cell distinguish the parental strand from the daughter strand in repairing replication errors,How does the cell restore the proper DNA sequence when the original sequence can no longer be read? How does the cell deal with lesions that block replication?,Topic 1: Replication errors and their rep

9、air,CHAPTER 9 The mutability and repair of DNA,How the replication errors are resulted? The nature of the replication errors is mismatch. 2. How mismatches are recognized and correctly repair?,The nature of mutations,Point mutations: Transitions (pyrimidine to pyrimidine, purine to purine) Transvers

10、ions (pyrimidine to purine, purine to pyrimidine),Replication errors and replication,Insertions Deletions Gross rearrangement of chromosome.,These mutations might be caused by insertion by transposon or by aberrant action of cellular recombination processes.,Rate of spontaneous mutation at any given

11、 site on chromosomal ranges from 10-6 to 10-11 per round of DNA replication, with some sites being “hotspot” . Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide sequences, known as DNA microsatellites (微卫星DNA). These sequences (1) are important in human genet

12、ics and disease, (2) hard to be copied accurately and highly polymorphic in the population.,How the replication errors are resulted?,Each bases has its preferred tautomeric form (Ch 6),The strictness of the rules for “Waston-Crick” pairing derives from the complementarity both of shape and of hydrog

13、en bonding properties between adenine and thymine and between guanine and cytosine.,Some replication errors escape proofreading,The 3-5 exonuclease activity of replisome only improves the fidelity of DNA replication by a factor of 100-fold. The misincorporated nucleotide needs to be detected and rep

14、laced, otherwise it will cause mutation (Fig. 9-2).,Replication errors and replication,Figure 9-2 Generation of Mutation,2. How the replication errors are repaired?,Mismatch repair removes errors that escape proofreading,Increase the accuracy of DNA synthesis for 2-3 orders of magnitudes. Two challe

15、nges: (1)rapidly find the mismatches/mispairs, (2) Accurately correct the mismatch,Replication errors and replication,Talking about the story of E. coli repair system,MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone MutS embraces the mismatch-containing

16、 DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself,Figure 9-4 Crystal structure of MutS,MutS is a dimer. One monomer interacts with the mismatch specifically, and the other nonspecifically.,DNA is kinked,MutS-mismatch-containing DNA complex recruits MutL, MutL act

17、ivates MutH, an enzyme causing an incision or nick on one strand near the site of the mismatch. Nicking is followed by the specific helicase (why?) (UrvD) and one of three exonucleases (why?).,DNA polymerase III,Helicase,Exonuclease,Detail 1: How does the E. coli mismatch repair system know which of

18、 the two mismatched nucleotide to replace?,The newly synthesized strand is not methylated by Dam methylase in a few minutes after the synthesis.,Figure 9-5,Detail 2: Different exonucleases are used to remove ssDNA between the nick created by MutH and the mismatch.,Figure 9-6,Eukaryotic cells also re

19、pair mismatches and do so using homologs to MutS (MSH) and MutL (MLH). The underlying mechanisms are not the same and not well understood.,Topic 2: DNA damage,CHAPTER 9 The mutability and repair of DNA,3/22/05,DNA undergoes damage spontaneously (自发的) from hydrolysis (水解) and deamination (转氨),Resulte

20、d from the action of water,DNA damage,Figure 9-7: Mutation due to hydrolytic damage,Deamination CU,Hydrolysis creates apurinic deoxyribose,Deamination 5-mC T,The presence of U and apurinic deoxyribose in DNA resulted from hydrolytic reactions is regarded as unnatural, thus is easily be recognized an

21、d repaired.,Can 5-mC T lesion be repaired?,Vertebrate DNA frequently contains 5-methyl cytosine in place of cytosine as a result of the action of methyl transferase. This modified base plays a role in the transcriptional silencing (Ch 17).,DNA is damaged by alkylation (烷基化), oxidation (氧化) and radia

22、tion (辐射),DNA damage,Figure 9-8 G modification,Alkylating chemical: Nitrosamines (亚硝胺),Reactive oxygen species (O2-, H2O2, OH),“O2-” hyperoxide “H2O2”Peroxide “OH” hydroxyl,Figure 9-9 Thymine dimer. UV induces a cyclobutane (环丁烷) ring between adjacent T.,Radiation damage 1,Gamma radiation and X-rays

23、 (ionizing radiation) cause double-strand breaks and are particularly hazardous (hard to be repaired).,Radiation damage 2,Mutations are also caused by base analogs (碱基类似物) and intercalating agents (嵌入剂),DNA damage,Base analogs: similar enough to the normal bases to be processed by cells and incorpor

24、ated into DNA during replication. But they base pair differently, leading to mispairing during replication. The most mutagenic base analog is 5-bromoUracil (5-BrU) (溴尿嘧啶).,Figure 9-10a Base analogues,Figure 3-33 G-U pair,烯醇异构体,酮异构体,Intercalating agents are flat molecules containing several polycycli

25、c rings that interact with the normal bases in DNA through hydrogen bonds and base stacking.,Figure 9-10b Intercalating agents,溴乙非啶,二氨基吖啶/原黄素,吖啶, 氮蒽,Topic 3: Repair of DNA damage,CHAPTER 9 The mutability and repair of DNA,3/22/05,Two consequence of DNA damage,Some damages, such as thymine dimer, nic

26、k or breaks in the DNA backbone, create impediments to replication or transcription Some damages creates altered bases that has no effect on replication but cause mispairing, which in turn can be converted to mutation.,Repair of DNA damage,Direct reversal of DNA damage by photoreactivation (光活化作用) a

27、nd alkyltransferase (烷基转移酶) Base excision repair (切割修复) Nucleotide excision repair Recombination (DSB) repairs Translesion DNA synthesis,Mechanisms to repair a damage,See Table 9-1 for summary,Repair of DNA damage,Direct reversal of DNA damage,Error-free repair,Repair of DNA damage,Photoreactivation,Figure 9-11,Monomerization of thymine dimers by DNA photolyases in the presence of visible light.,Methyltransferase,Removes the methyl group from the me