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

In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoen-zyme. This complex includes the polymerase and proofreading subunits, the processivity clamp and clamp loader complex. The holC gene encodes an ac-cessory protein (known as chi) 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 sensi-tivity to DNA damaging agents. In this study we isolate spontaneous suppressor mutants in a holC increment 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 affect-ing transcription elongation and termination for syner-gistic or suppressive effects on holC mutant pheno-types. Alleles of RpoA (alpha), RpoB (beta) and RpoC (beta') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mu-tants. 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 com-plexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA func-tion are particularly important to sustain viability and chromosome integrity.