《分子生物学》4cha

1、,Chapter 9:,Chapter 9:,The mutability and repair of DNA,Causes Repair,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-pairing DNA polymorase I,The nature of mutatio

2、ns,Point 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,Po

3、int mutation,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

4、challenges:,Rapidly find the mismatches/mispairs 2 Accurately correct the mismatch,Prokaryote Mismatch repair system,MutS MutL MutH UrvD one 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

5、, inducing a pronounced 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

6、the mismatch.,Nicking is 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

7、mismatch.,Nicking is followed 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,Repli

8、cation generate hemimethylation 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

9、cells also repair mismatches and 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 apuri

10、nic deoxyribose,Deamination 5-mC T,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 di

11、mer, nick 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.,DNA damage and their repair,Causes,Repair,Direct reversal of DNA damage,P

12、hotoreactivation,Monomerization of thymine dimers 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,A

13、P endonulease & exonulcease Cleaves the abasic sugars DNA polymerase/ligase Works sequentially to complete the repair event.,Glycosylase,Recognizes the damaged base Removes the damaged base,oxoG:A repair. A glycosylase recognizes the mispair and removes A.,Fail-safe systems,Nucleotide Excision repai

14、r,Recognize distortions to the shape of the DNA double helix Remove 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 DNA Uvr B: melt DNA to create ssDNA recruits C Uvr C create two incisions Uv

15、r D release the ssDNA,Transcription-couple repair: nucleotide,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 repair 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 DN