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The role of replication clamp-loader protein HolC of Escherichia coli in overcoming replication / transcription conflicts

Deani L. Cooper, Taku Harada, Samia Tamazi, Alexander E. Ferrazzoli, View ORCID ProfileSusan T. Lovett
doi: https://doi.org/10.1101/2020.12.02.408393
Deani L. Cooper
1Department of Biology and Rosenstiel Basic Medical Sciences Research Center Brandeis University, MS029, Waltham, MA 02453
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Taku Harada
1Department of Biology and Rosenstiel Basic Medical Sciences Research Center Brandeis University, MS029, Waltham, MA 02453
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Samia Tamazi
1Department of Biology and Rosenstiel Basic Medical Sciences Research Center Brandeis University, MS029, Waltham, MA 02453
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Alexander E. Ferrazzoli
1Department of Biology and Rosenstiel Basic Medical Sciences Research Center Brandeis University, MS029, Waltham, MA 02453
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Susan T. Lovett
1Department of Biology and Rosenstiel Basic Medical Sciences Research Center Brandeis University, MS029, Waltham, MA 02453
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  • ORCID record for Susan T. Lovett
  • For correspondence: lovett@brandeis.edu
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ABSTRACT

In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits as well as the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as x) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study, to elucidate the role of HolC in replication, we isolate spontaneous suppressor mutants in a holCΔ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains or sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β) and RpoC (β’) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. Mfd had no effect nor did elongation factors GreA and GreB. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA rpoC and sspA.These results are consistent with the hypothesis that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.

IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of E. coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps resolve codirectional collisions between replication and transcription complexes, which become toxic in HolC’s absence.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵† Alexander Ferrazzoli passed away on August 4, 2020.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 02, 2020.
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The role of replication clamp-loader protein HolC of Escherichia coli in overcoming replication / transcription conflicts
Deani L. Cooper, Taku Harada, Samia Tamazi, Alexander E. Ferrazzoli, Susan T. Lovett
bioRxiv 2020.12.02.408393; doi: https://doi.org/10.1101/2020.12.02.408393
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The role of replication clamp-loader protein HolC of Escherichia coli in overcoming replication / transcription conflicts
Deani L. Cooper, Taku Harada, Samia Tamazi, Alexander E. Ferrazzoli, Susan T. Lovett
bioRxiv 2020.12.02.408393; doi: https://doi.org/10.1101/2020.12.02.408393

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