DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts

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, the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as χ) 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 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, and of 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. 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. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose 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.

Keywords: DNA polymerase; RNA polymerase; Rho; transcription termination.

Figure 1. FIGURE 1: Events leading to fork breakage after collision of DNA pol III with transcription elongation complexes (TEC).

Diagrammed are events ensuing from codirectional collisions, but can occur similarly with head-on collisions. RNAP is illustrated in red, the fork helicase in green. (A) The fork encounters a TEC, that blocks DNA polymerization. (B) The fork moves on, replication is restarted. (C) A single-strand gap is left behind. (D) Convergence of a second fork (helicase marked with lt green) onto the gap. (E) The resulting one-ended double-strand break.

Figure 2. FIGURE 2. (A)

Head-on collisions between the replication fork and transcription complexes. Because the DnaB fork helicase (green) translocates on the lagging strand template, head-on collisions with RNAP (red) blocks fork progression. (B) Codirectional collisions. DnaB fork helicase can proceed unimpeded, but fork unwinding can stabilize extensive R-loops (RNA/DNA hybrids) behind RNAP. Copyright © American Society for Microbiology, mBio12, 2021, e00184-21 doi: 10.1128/mBio.00184-21