Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Abstract

Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging strand template coated with Single-Stranded DNA-Binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C-terminus of SSB. In vitro, these interactions trigger re-modeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB re-modeling reaction in vitro. Plasmid-encoded priC351(R155A) did not complement priC303::kan and priA341(R697A) has not yet been tested for complementation. Here, we further study the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E) and priC351(R155A) on the chromosome and characterizing these mutant strains. All three priA mutants behaved as wild type. In a ΔpriB strain, they caused modest increases in SOS expression, cell size and defects in nucleoid partitioning (Par-). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). priC351(R155A) behaved as expected: no phenotype as a single mutant and displayed severe growth defects when combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous, Par- and had high SOS expression. We conclude that in vivo priC351(R155A) identified an essential residue and function for PriC and that PriA R697 and Q701 are only important in the absence of PriB and that region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C-terminus of SSB interact with certain residues in PriA and PriC to recruit those proteins to the repaired fork where they help re-model it for restart. Here, we place those mutations on the chromosome and test the effect of mutating these residues in vivo. The priC mutation completely abolishes function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.