A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

The initiation of replication in bacteria is regulated via the initiator protein DnaA. ATP-bound DnaA binds to multiple sequences at the origin of replication, oriC, unwinding the DNA and promoting the binding of DnaB helicase. From an Escherichia coli mutant highly perturbed for replication control, obgE∷Tn5-EZ seqAΔ, we isolated multiple spontaneous suppressor mutants with enhanced growth and viability. These suppressors suppressed the replication control defects of mutants in seqA alone and genetically mapped to the essential dnaA replication initiator gene. DNA sequence analysis of four independent isolates revealed an identical deletion of the DnaA-coding region at a repeated hexanucleotide sequence, causing a loss of 25 amino acids in domain II of the DnaA protein. Previous work has established no function for this region of protein, and deletions in the region, unlike other domains of the DnaA protein, do not produce lethality. Flow cytometric analysis established that this allele, dnaAΔ96-120, ameliorated the over-replication phenotype of seqA mutants and reduced the DNA content of wild-type strains; virtually identical effects were produced by loss of the DnaA-positive regulatory protein DiaA. DiaA binds to multiple DnaA subunits and is thought to promote cooperative DnaA binding to weak affinity DNA sites through interactions with DnaA in domains I and/or II. The dnaAΔ96-120 mutation did not affect DiaA binding in pull-down assays, and we propose that domain II, like DiaA, is required to promote optimal DnaB recruitment to oriC

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

<div><p>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 <i>E</i>. <i>coli</i> genetic system, for which replication and repair factors have been well-defined. By using high-expression suppressor screens, we identified <i>yoaA</i>, encoding a putative helicase, and <i>holC</i>, 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; <i>E</i>. <i>coli</i> has a paralog to YoaA, DinG, which possesses 5’ to 3’ helicase activity and an Fe-S cluster essential to its activity. Mutants in <i>yoaA</i> are sensitive to AZT exposure; <i>dinG</i> mutations cause mild sensitivity to AZT and exacerbate the sensitivity of <i>yoaA</i> mutant strains. Suppression of AZT sensitivity by <i>holC</i> or <i>yoaA</i> 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.</p></div

Survival of <i>holC</i>, <i>yoaA</i> and <i>dinG</i> mutants to AZT.

<p>(A) Plating of MG1655 (wild-type), <i>yoaA</i>, <i>yoaA dinG</i>, <i>dinG</i>, <i>ΔholC</i>::<i>kan</i>, or <i>ΔholC</i>::<i>kan yoaA</i> on LB medium with or without AZT. Ten-fold serial dilutions are shown from cultures grown and plated in parallel. Dilutions of other strains not relevant to this analysis were cropped out of these images. (B) Fractional survival of MG1655 (wild-type, open square), <i>yoaA</i> (open down triangle), <i>dinG</i> (open circle), or <i>yoaA dinG</i> (open diamond) mutant strains. (C) Fractional survival of MG1655 (wild-type, open square), <i>yoaA</i> (open down triangle), <i>holC</i> (open up triangle), or <i>yoaA holC</i> (open circle) mutant strains. Ten-fold serial dilutions of the indicated strains were plated on LB medium with or without AZT. Data represent n = 4 to n = 8 independent single colony isolates, shown as the average and standard deviation of log₁₀-transformed fractional survivals.</p

Survival of <i>holC</i>, <i>yoaA</i> and <i>yoaA dinG</i> mutants to AZT.

<p>(A) Fractional survival of <i>holCΔ</i>::<i>kan</i>, <i>yoaA</i>, or <i>yoaA dinG</i> mutants carrying the following plasmids: pNTR-Control, (closed square, black line); pNTR-HolC, (closed up triangle, blue line); or pNTR-YoaA, (closed down triangle, red line). (B) Survival of <i>holCΔ</i>::<i>kan yoaA</i>, or <i>yoaA dinG</i> mutants carrying the following plasmids: pNTR-Control, pNTR-HolC, or pNTR-YoaA. Ten-fold serial dilutions of the indicated strains were plated on LB medium with or without AZT. Data represent n = 6 to n = 8 independent single colony isolates, shown as the average and standard deviation of log₁₀-transformed fractional survivals. Representative images of plates are shown.</p

Survival to AZT of wild type and <i>yoaA</i> mutants expressing mutant YoaA on mobile plasmids.

<p>(A) Fractional survival of MG1655 (wild-type) or <i>yoaA</i> carrying the following plasmids: pNTR-Control, (square, black line); pNTR-YoaA, (down triangle, red line); pNTR-YoaA K51A, (hexagon, orange line); pNTR-YoaA D225A, (circle, purple line); or pNTR-YoaA C168S, (diamond, blue line). (B) Survival of MG1655 (wild-type) or <i>yoaA</i> carrying the following plasmids: pNTR-Control, pNTR-YoaA, pNTR-YoaA K51A, pNTR-YoaA D225A, or pNTR-YoaA C168S. Ten-fold serial dilutions of the indicated strains were plated on LB medium with or without AZT. Data represent n = 4 independent single colony isolates, shown as the average and standard deviation of log₁₀-transformed fractional survivals. Representative images of plates are shown. Rows were from the same batch of plates in an assay done on the same day, rows of other strains not relevant to this figure were cropped out of these images.</p

Survival of <i>lexA3</i>, <i>recA</i> and <i>xthA</i> mutants to AZT.

<p>(A) Fractional survival of MG1655 (wild-type) carrying the following plasmids: pNTR-Control, (closed square, black line); pNTR-HolC, (closed up triangle, blue line); or pNTR-YoaA, (closed down triangle, red line). (B) Fractional survival of <i>lexA3</i> carrying the following plasmids: pNTR-Control, (closed square, black line); pNTR-HolC, (closed up triangle, blue line); or pNTR-YoaA, (closed down triangle, red line). (C) Fractional survival of <i>recA</i> mutants carrying the following plasmids: pNTR-Control, (closed square, black line); pNTR-HolC, (closed up triangle, blue line); or pNTR-YoaA, (closed down triangle, red line). (D) Fractional survival of <i>xthA</i> carrying the following plasmids: pNTR-Control, (closed square, black line); pNTR-HolC, (closed up triangle, blue line); or pNTR-YoaA, (closed down triangle, red line). (E) Survival to AZT of MG1655 (wild type), <i>lexA3</i>, <i>recA</i>, or <i>xthA</i> carrying the following plasmids: pNTR-Control, pNTR-HolC, or pNTR-YoaA. Ten-fold serial dilutions of the indicated strains were plated on LB medium with or without AZT. Data represent n = 6 to n = 8 independent single colony isolates, shown as the average and standard deviation of log₁₀-transformed fractional survivals. Representative images of plates are shown.</p

Survival to AZT of wild type and <i>holC</i> mutants expressing mutant HolC on mobile plasmids.

<p>(A) Fractional survival of MG1655 (wild-type) or <i>holCΔ</i>::<i>kan</i> mutant strains carrying the following plasmids: pNTR-Control, (square, black line); pNTR-HolC, (up triangle, blue line); pNTR-HolC F64A, (star, orange line); pNTR-HolC Y131L, (hexagon, green line); pNTR-HolC V117F, (circle, purple line); or pNTR-HolC R128A, (diamond, light blue line). (B) Survival of MG1655 (wild-type) or <i>holC</i> mutant strains carrying the following plasmids: pNTR-Control, pNTR-HolC, pNTR-HolC F64A, pNTR-HolC V117F, pNTR-HolC R128A, pNTR-HolC Y131L. Ten-fold serial dilutions of the indicated strains were plated on LB medium with or without AZT. Data represent n = 3 independent single colony isolates, shown as the average and standard deviation of log₁₀-transformed fractional survivals. Representative images of plates are shown.</p