SeqA structures behind Escherichia coli replication forks affect replication elongation and restart mechanisms

Abstract

The SeqA protein binds hemi-methylated GATC sites and forms structures that sequester newly replicated origins and trail the replication forks. Cells that lack SeqA display signs of replication fork disintegration. The broken forks could arise because of over-initiation (the launching of too many forks) or lack of dynamic SeqA structures trailing the forks. To confirm one or both of these possible mechanisms, we compared two seqA mutants with the oriCm3 mutant. The oriCm3 mutant over-initiates because of a lack of origin sequestration but has wild-type SeqA protein. Cells with nonfunctional SeqA, but not oriCm3 mutant cells, had problems with replication elongation, were highly dependent on homologous recombination, and exhibited extensive chromosome fragmentation. The results indicate that replication forks frequently break in the absence of SeqA function and that the broken forks are rescued by homologous recombination. We suggest that SeqA may act in two ways to stabilize replication forks: (i) by enabling vital replication fork repair and restarting reactions and (ii) by preventing replication fork rear-end collisions.

Phenotypes of dnaX E145A Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks

“…At permissive temperature, the mutant cells typically contained from about three to nine chromosomes, whereas at the nonpermissive temperature, the chromosome content was reduced to between zero and four. The recA(Ts) single mutant also exhibited chromosome loss, as reported previously (36,49), but to a much lesser extent than the double mutant (seen as more cells in the 3-chromosome peak and fewer cells in the 4-chromosome peak than at permissive temperature) ( Fig. 4, row 2).…”

“…Cultures of cells growing exponentially (to an OD of ϳ0.15) at 30°C were split, and rifampin (450 g/ml) and cephalexin (10 g/ml) both were added to each culture. One portion was kept at the permissive temperature (30°C), whereas the other was shifted to the nonpermissive temperature (42°C) to determine the loss RecA function (36,49). Cells were harvested before drug treatment and after 3 to 4 generation times in the presence of the drugs and fixed for analysis with flow cytometry to compare the replication fork run-out at each temperature.…”

A genetic screen to identify factors affected by undecaprenyl phosphate recycling uncovers novel connections to morphogenesis inEscherichia coli

“…Thus, it is unlikely that SulA is soley responsible for the shape defect of Δ3PP lon::mini-Tn10 cells at 42°C. Mutations that affect seqA (Lu et al, 1994, Pedersen et al, 2017 or rep (Michel et al, 1997) lead to double-stranded DNA breaks, which activates the SOS response (Ossanna & Mount, 1989, Rotman et al, 2014, triggering sulA expression and thus filamentation ( Figure S2). The Min proteins MinC, MinD, and MinE function to restrict FtsZ ring formation to midcell (de Boer et al, 1989).…”

Replication Fork Breakage and Restart in Escherichia coli

“…In parallel with studies dedicated to replication restart, several investigations have aimed to understand how replication impairment can lead to the formation of dsDNA ends at forks. Although blocked forks might be inherently fragile owing to their ssDNA regions, only the seqA mutant was proposed to suffer direct breakage of ssDNA at stalled replication forks (24,25), and it turned out that most often, arrested forks were not broken. Three main modes of dsDNA end formation at forks were reported: (i) encounter of a replication fork with a preexisting single-stranded DNA interruption in a template strand (originally called "replication fork collapse" in a seminal review by A. Kuzminov [26]) (Fig.…”