Alternative complexes formed by the Escherichia coli clamp loader accessory protein HolC (x) with replication protein HolD (ψ) and repair protein YoaA

• Published in: DNA repair Vol. 100; p. 103006
• Main Authors: Sutera, Vincent A., Weeks, Savannah J., Dudenhausen, Elizabeth E., Baggett, Helen B. Rappe, Shaw, McKay C., Brand, Kirsten A., Glass, David J., Bloom, Linda B., Lovett, Susan T.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2021
• Subjects: DNA helicase; DNA polymerase; DNA Polymerase III - metabolism; DNA Repair; DNA Replication; DNA, Bacterial - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Protein Binding; Protein Conformation; SSB; DNA helicase; DNA replication; SSB; DNA polymerase; AZT; DNA repair; Y2H
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2020.103006

[Display omitted] •HolC binds to two exclusive partners, the clamp-loader protein, HolD or the repair helicase, YoaA.•HolC residues W57 and F64 are required for interaction with YoaA as well as HolD.•These HolC residues do not affect its binding to single-strand DNA binding protein, SSB.•HolC can recruit alternative complexes to balance replication vs. repair reactions at the replication fork. Efficient and faithful replication of DNA is essential for all organisms. However, the replication fork frequently encounters barriers that need to be overcome to ensure cell survival and genetic stability. Cells must carefully balance and regulate replication vs. repair reactions. In Escherichia coli, the replisome consists of the DNA polymerase III holoenzyme, including DNA polymerase, proofreading exonuclease, processivity clamp and clamp loader, as well as a fork helicase, DnaB and primase, DnaG. We provide evidence here that one component of the clamp loader complex, HolC (or χ) plays a dual role via its ability to form 2 mutually exclusive complexes: one with HolD (or ψ) that recruits the clamp-loader and hence the DNA polymerase holoenzyme and another with helicase-like YoaA protein, a DNA-damage inducible repair protein. By yeast 2 hybrid analysis, we show that two residues of HolC, F64 and W57, at the interface in the structure with HolD, are required for interaction with HolD and for interaction with YoaA. Mutation of these residues does not interfere with HolC’s interaction with single-strand DNA binding protein, SSB. In vivo, these mutations fail to complement the poor growth and sensitivity to azidothymidine, a chain-terminating replication inhibitor. In support of the notion that these are exclusive complexes, co-expression of HolC, HolD and YoaA, followed by pulldown of YoaA, yields a complex with HolC but not HolD. YoaA fails to pulldown HolC-F64A. We hypothesize that HolC, by binding with SSB, can recruit the DNA polymerase III holoenzyme through HolD, or an alternative repair complex with YoaA helicase.