The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming Replication/Transcription Conflicts

In Escherichia coli, DNA replication is catalyzed by an assembly of pro-teins, 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 x) to the core clamp loader com-plex and is the only protein of the holoenzyme that binds to single-strand DNA bind-ing 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 DholC strain and identify these by whole-genome sequencing. Some suppressors are alleles of RNA polymerase, suggest -ing that transcription is problematic for holC mutant strains, or alleles of sspA, encod-ing 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 (a), RpoB (/3), and RpoC (/39) RNA poly-merase holoenzyme can partially suppress loss of HolC. In contrast, mutations in tran-scription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-de-pendent 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 that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.