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.

Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

“…This damage could simply be the continued association of the nuclease with its site of action, resulting in a polymerase roadblock. Candidates for repair processing include primase ( priA ), involved in replisome assembly at stalled forks ( 49 ); DnaK, required for RecA-independent replication fork repair ( 50 ); or YoaA, a helicase thought to assist with the removal of blocked termini by displacement of the primer terminus ( 51 ). Single-strand annealing processes to promote primer-template switching (as observed in Salmonella [ 52 ]) and inducible RecA-independent repair mechanisms (such as the RpoS-mediated response [ 53 ]) could also participate in recombinant formation.…”

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication

“…A new, well-conserved bacterial protein YoaA, was identified in a genetics screen to be involved in coordinating repair and replication machinery at blocked replication forks. Based on sequence similarity to DinG, YoaA was predicted to be a [4Fe4S] protein (85).…”

Omnipresent Maxwell's demons orchestrate information management in living cells

“…Lhr(RhlF) is a large helicase of unknown specificity also active in M. tuberculosis, possibly involved in site-specific recombination . HelC(YoaA) directs repair factors to a blocked DNA fork (Brown et al, 2015). HelG is a paralogue, whose ATPase activity is stimulated by single-stranded DNA (Cheng and Wigley, 2018).…”