Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein

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.

正在延伸的DNA聚合酶常会遭遇阻碍其行进的DNA损伤或结构，且需在DNA复制完成前完成修复。因此，将修复因子定向招募至停滞的复制叉，且不干扰正常复制过程，对维持细胞正常功能至关重要，而这一过程的分子机制目前尚未被充分阐明。为研究该过程，我们采用了链终止型核苷类似物3'-叠氮胸苷（3’ azidothymidine, AZT）以及遗传背景清晰的大肠杆菌（E. coli）遗传系统，该系统的复制与修复因子已得到充分鉴定。通过高表达抑制筛选，我们鉴定出两个可提升菌株对AZT耐受性的基因：编码推定解旋酶的yoaA，以及编码复制钳加载器Chi亚基的holC。YoaA属于XPD/RAD3家族的推定铁硫簇解旋酶，其直系同源基因广泛存在于多数细菌基因组中；大肠杆菌自身还存在YoaA的旁系同源蛋白DinG，该蛋白具备5'→3'解旋酶活性，且其活性依赖于铁硫簇。yoaA突变体对AZT暴露敏感，而dinG突变体仅表现出轻度AZT敏感性，且会加剧yoaA突变菌株的敏感表型。holC或yoaA对AZT敏感性的抑制作用存在相互依赖关系，同时我们的实验证据表明YoaA与Chi存在物理相互作用。Chi与单链DNA结合蛋白（single-strand DNA binding protein, SSB）以及Psi的相互作用，与校读型3'核酸外切酶DnaQ一样，均对提升AZT耐受性不可或缺。我们的研究表明，修复过程可通过上述相互作用与停滞的复制过程相偶联。我们提出假说：SSB通过Chi将YoaA解旋酶招募至复制间隙，新生链的解旋可促进修复过程与AZT的切除。这种招募机制可避免解旋酶活性产生毒性，并协助修复因子与复制因子完成交接，从而确保复制相关修复及时完成并恢复复制进程。