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1、,Chapter 9:,Chapter 9:,The mutability and repair of DNA,CausesRepair,Mutation,Recombination,Homologous recombination(同源重组）Site-specific recombination(位点专一重组)Transposition(转座）,The causes of mutation,Mutagens,Error in replication,tautomerism on base-pairingDNA polymorase I,The nature of mutations,Poin
2、t mutations:Transitions(pyrimidine to pyrimidine,purine to purine)Transversions(pyrimidine to purine,purine to pyrimidine),Insertions or deletions,ATG,GTG,GAG,CCG,GCC,GAG,TAG,ATG,GTG,GAG,CTG,GCC,GAG,TAG,ATG,GTG,GAG,CCG,GCC,GAG,TAG,ATG,GTG,GAG,CG,GCC,GAG,TAG,ATG,GTG,GAG,CGG,CCG,AGT,AG,Point mutation,
3、Insertions or deletions,missense,Errors in replication,Replication errors and their repair,Repair of Replication errors:Mismatch repair Increase the accuracy of DNA synthesis for 2-3 orders of magnitudes.,Prokaryote Eukaryote,Mismatch repair removes errors that escape proofreading,Two challenges:,Ra
4、pidly find the mismatches/mispairs2 Accurately correct the mismatch,Prokaryote Mismatch repair system,MutSMutLMutHUrvDone of three exonucleases,MutS scans the DNA,recognizing the mismatch from the distortion they cause in the DNA backbone MutS embraces the mismatch-containing DNA,inducing a pronounc
5、ed kink in the DNA and a conformational change in MutS itself,DNA is kinked,MutS is a dimer.One monomer interacts with the mismatch specifically,and the other nonspecifically.,recruits MutL,MutL activates MutH,enzyme causing an incision or nick on one strand near the site of the mismatch.,Nicking is
6、 followed by the specific helicase(UrvD)and one of three exonucleases,Different exonucleases are used to remove ssDNA between the nick created by MutH and the mismatch.,recruits MutL,MutL activates MutH,enzyme causing an incision or nick on one strand near the site of the mismatch.,Nicking is follow
7、ed by the specific helicase(UrvD)and one of three exonucleases,Exonuclease VII or I,DNA polymerase III,How does the E.coli mismatch repair system know which of the two mismatched nucleotide to replace?,DAM methylase methylate A residues on both strands of 5-GATC-3,Replication generate hemimethylatio
8、n DNA in E.coli,MutH makes incision unmethylated daughter strand,Errors in replication,Replication errors and their repair,Repair of Replication errors:Mismatch repair Increase the accuracy of DNA synthesis for 2-3 orders of magnitudes.,Prokaryote Eukaryote,Eukaryotic cells also repair mismatches an
9、d do so using homologs to MutS(MSH)and MutL(MLH).The underlying mechanisms are not the same and not well understood.,DNA damage and their repair,Causes,Repair,DNA undergoes damage spontaneously from hydrolysis and deamination,Deamination CU,Hydrolysis creates apurinic deoxyribose,Deamination 5-mC T,
10、Explaining why DNA contains T instead of U,Alkylation,Oxidation,Nitrosamines(亚硝胺),Reactive oxygen species(O2-,H2O2,OH),Radiation,base analogspoint mutation intercalating agentsinsertion or deletion,Two consequence of DNA damage,Some damages,such as thymine dimer,nick or breaks in the DNA backbone,cr
11、eate impediments to replication or transcriptionSome damages creates altered bases that has no effect on replication but cause mispairing,which in turn can be converted to mutation.,DNA damage and their repair,Causes,Repair,Direct reversal of DNA damage,Photoreactivation,Monomerization of thymine di
12、mers by DNA photolyases in the presence of visible light.,Methyltransferase catalyzes the transfer of the methyl group on O6-methylguanine to cystein residue on the enzyme,thereby restoring the normal G in DNA.,Methyl group removal,Base Excision repair,AP endonulease&exonulcease Cleaves the abasic s
13、ugarsDNA polymerase/ligase Works sequentially to complete the repair event.,Glycosylase,Recognizes the damaged baseRemoves the damaged base,oxoG:A repair.A glycosylase recognizes the mispair and removes A.,Fail-safe systems,Nucleotide Excision repair,Recognize distortions to the shape of the DNA dou
14、ble helixRemove a short single-stranded segment that includes the lesion.DNA polymerase/ligase fill in the gap.,Nucleotide Excision-Repair,Uvr A:detect the distortion of DNAUvr B:melt DNA to create ssDNA recruits CUvr C create two incisionsUvr D release the ssDNA,Transcription-couple repair:nucleoti
15、de,Excision repair system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template,Recombination repairs,Double-strand break(DSB)repair pathway,Figure 10-4.Damage in the DNA template can lead to DSB formation during replication,FIGURE 10-3 DSB repai
16、r model for homologous recombination,Translesion DNA synthesis,Occurs when the above repairs are not efficient enough so that a replicating polymerase encounters a lesion,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 damageTranslesion polymerases are expressed as part of the SOS response pathway.,Translesion DNA synthesis in E.coli,
