engleski

Mismatch repair in eukaryotic DNA is not strand-directed by methylation

Mismatched base pairs in DNA arise from replication errors, from 5-methyl-cytosine deamination and from recombination that results in the pairing of homologous but nonidentical sequences. An organism may respond to these mispairs and convert them to the normal base pairs or may ignore them and produce nonidentical and mutated progeny, as well as modified transcripts. If the cell chooses to repair a mismatch it is faced with the problem how to recognize and specifically eliminate the strand bearing the noncorrect sequence. There is a lot of convincing evidence that eukaryotic cells (from yeasts to mammalian cells) possess very potent mismatch repair activity. It was shown that a nick or gap in the vicinity of mismatch stimulates the excision of mispaired base but the mechanism for strand discrimination is still unclear. A major role in directing correction of the replication errors in E. coli is a methylation of adenine in the dGATC sequence located less than a kilobase away. Newly synthesized, temporarily unmethylated strand carrying mismatched bases is targeted by mismatch recognition and excision enzymes (MutS,L,H, helicase II). The reaction responsible for strand-specific correction of mispaired bases has been highly conserved during evolution, and homologues of bacterial MutS and MutL have been identified in some eukaryots including man. Inactivation of genes encoding these activities results in the large increase in spontaneous mutability and in the hereditary predisposition to tumor development. The only known type of methylation in eukaryotic cells is that of inner C in the dCCGG sequence. Hpa II methylase methylates the same sequence in the same way, and we used it for in vitro methylation of DNA. Covalently closed circular heteroduplexes carrying TˇG or CˇA mismatched base pairs, were constructed in vitro by annealing DNAs from two mutants of phage M13. Hemi-methylated or fully-methylated heteroduplexes were exposed to Xenopus laevis egg extracts. We found that: i) extracts possessed very high repair activity; correction efficiency was 108 repair events per egg equivalent; ii) correction in favor of C:G base pair generation occurred with almost equal efficiency for the TˇG and CˇA mispairs; iii) there was no significant difference in repair efficiency for mismatched bases in hemi-methylated and fully-methylated heteroduplexes indicating that the repair of mismatched bases in X. laevis egg extracts was not strand-directed by the methylation of DNA.

nije evidentirano

nije evidentirano