title: Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein.
abstract: Elongating DNA polymerases frequently encounter lesions or structures that impede progress and require repair before DNA replication can be completed. Therefore, directing repair factors to a blocked fork, without interfering with normal replication, is important for proper cell function, and it is a process that 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. By using high-expression suppressor screens, 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. 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. Suppression of AZT sensitivity by holC or yoaA was mutually codependent and we provide evidence here that YoaA and Chi physically interact. Interactions of Chi with single-strand DNA binding protein (SSB) and with Psi were required to aid AZT tolerance, as was the proofreading 3' exonuclease, DnaQ. Our studies suggest that repair is coupled to blocked replication through these interactions. We hypothesize that SSB, through Chi, recruits the YoaA helicase to replication gaps 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.
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America.
authors.forename: Laura T
authors.initials: LT
authors.lastname: Brown
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America.
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Sutera
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America.
authors.forename: Shen
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Weitzel
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America.
authors.forename: Yisha
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America.
authors.forename: Susan T
authors.initials: ST
authors.lastname: Lovett
mesh_terms: D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D015215:Zidovudine / Q000494:pharmacology*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D004259:DNA-Directed DNA Polymerase