Throughout the growth cycle, cells are faced with a delicate balancing act between DNA damage response and replication, transcription or translation. Frequently encountered lesions or damage can impede or stall replication forks and require repair before DNA replication can be completed. Directing repair factors to a blocked fork, without interference with normal replication, is therefore important for proper cell function, but is not well understood. To study this process, we have employed the chain-terminating nucleoside analog, 3’ azidothymidine (AZT) and the E. coli genetic system, for which replication and repair factors have been well defined. Using high-expression suppressor screens in E. coli, we identified yoaA , encoding a putative helicase, and holC, encoding the Chi component of the replication clamp loader, as genes that promoted tolerance to AZT exposure. Suppression by holC or yoaA was mutually codependent and we provide evidence here that YoaA and Chi physically interact. Our studies indicate that YoaA has ATPase activity. YoaA is a putative Fe-S helicase in the XPD/RAD3 family for which orthologs can be found in most bacterial genomes; E. coli has a paralog to YoaA, DinG, which possesses 5’ to 3’ helicase activity and an Fe-S cluster essential to its activity. Mutants in yoaA are sensitive to AZT exposure; dinG mutations cause mild sensitivity to AZT and exacerbate the sensitivity of yoaA mutant strains. Interactions of Chi with single-strand DNA binding protein (SSB) and Psi were required to aid AZT tolerance, as was the proofreading 3’ exonuclease, DnaQ. Furthermore, we provide evidence that YoaA is involved in the SOS response, in a LexA and RecA-dependent manner, and that its putative 16-22 base pair LexA binding box in the 5’-upstream promoter region is functionally required for this response. Our studies suggest that repair is coupled to blocked replication through these interactions. We hypothesize that SSB, through Chi, recruits the YoaA helicase to the fork and that unwinding of the nascent strand promotes repair and AZT excision. This recruitment prevents the toxicity of helicase activity and aids the handoff of repair with replication factors, ensuring timely repair and resumption of replication.
|Commitee:||Haber, James, Hedstrom, Lizbeth, McVey, Mitch|
|Department:||Molecular and Cell Biology|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 77/02(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Azidothymidine, Clamp loader, DNA replication, Helicase|
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