The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming 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 ΔholC 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 alleles of sspA, encoding 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 that 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 Escherichia 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 avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC's absence.

Keywords: DNA repair; DNA replication; stringent response; transcription factors.

FIG 1

Suppression of holC by sspA. Tenfold serial dilutions of cultures with and without the holC complementing plasmid (+pAM34-holC and −pAM34-holC, respectively) were plated on minimal glucose (min), minimal glucose Casamino Acids (min CAA), or LB medium and incubated at 30°C, 37°C, and 42°C, as indicated.

FIG 2

Violin plot of the cell length distribution of holC mutant derivatives grown in min CAA at 30°C as determined by microscopy. The dashed lines indicate the median values and dotted lines the quartile values.

FIG 3

Suppression of holC by SOS-induced functions. Tenfold serial dilutions of cultures cured for the holC complementing plasmid (pAM34-holC) were plated on minimal glucose (min) medium and incubated at 30°C, 37°C, and 42°C, as indicated.

FIG 4

Suppression of holC by rpoA dup(aa179-186). Tenfold serial dilutions of cultures with and without the holC complementing plasmid (+pAM34-holC and −pAM34-holC, respectively) were plated on minimal glucose (min), minimal glucose Casamino Acids (min CAA), or LB medium and incubated at 30°C, 37°C, and 42°C, as indicated.

FIG 5

Suppression of holC by rpoC::GFP. Tenfold serial dilutions of cultures without the holC complementing plasmid were plated on minimal glucose (min), minimal glucose Casamino Acids (min CAA), or LB medium and incubated at 30°C, 37°C, and 42°C, as indicated.

FIG 6

Enhancement of holC growth defects by dksA. Tenfold serial dilutions of cultures with and without the holC complementing plasmid (+pAM34-holC and −pAM34-holC, respectively) were plated on minimal glucose Casamino Acids (min CAA) or LB medium and incubated at 37°C and 42°C, as indicated.

FIG 7

Enhancement of holC growth defects by nusA11. Tenfold serial dilutions of cultures with and without the holC complementing plasmid (+pAM34-holC and −pAM34-holC, respectively) were plated on minimal glucose Casamino Acids (min CAA) or LB medium and incubated at 37°C and 42°C, as indicated. These strains are derived from AB1157 (ΔRac) in which lethal effects of nusA mutations are reduced.

FIG 8

Suppression of holC growth defects by rpoB8 and enhancement by rpoB3370. Tenfold serial dilutions of cultures with and without the holC complementing plasmid (+pAM34-holC and −pAM34-holC, respectively) were plated on minimal glucose Casamino Acids (min CAA) or LB medium and incubated at 37°C and 42°C, as indicated.

FIG 9

Bicyclomycin (BCM) sensitivity of holC mutants and holC suppression. Tenfold serial dilutions of cultures cured of the holC complementing plasmid were plated on minimal glucose Casamino Acids (min CAA) without and with two doses of bicyclomycin. (A) The rpoA allele is rpoA dup(aa179-186) and the sspA allele is a deletion. Suppression of holC by these alleles is reduced or abolished at 30°C on min CAA medium. (B) The rpoC allele is the rpoC::GFP allele, which partially suppresses holC on min CAA at 30°C and 37°C but not in the presence of bicyclomycin.

FIG 10

Enhancement of holC growth defects by a chromosomal inversion of rrnA, Inv A. Tenfold serial dilutions of cultures with and without the holC complementing plasmid (+pAM34-holC and −pAM34-holC, respectively) were plated on minimal glucose Casamino Acids (min CAA) or LB medium and incubated at 30°C, 37°C, and 42°C, as indicated. Inv A strains are plated in parallel to isogenic strains lacking the inversion (“control”). Two independent isolates were plated for the ΔholC derivatives.

FIG 11

Transcription/replication conflicts. DnaB fork helicase is illustrated in green, RNA polymerase is in red, DNA is in black, and RNA is in blue. (A) Head-on collisions lead to fork arrest (B) Codirectional collisions cause uncoupling of leading and lagging strand synthesis and possible stabilization of R-loops.