RecA is essential for Double-Strand Break Repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and member of the Structural Maintenance of Chromosomes (SMC) family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ΔrecN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ΔrecN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS, Rec− , had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain reflecting greater stringency of RecA's role in that background.

In Escherichia coli, after DNA damage, the SOS response increases the transcription (and protein levels) of approximately 50 genes. As DNA repair ensues, the level of transcription returns to homeostatic levels. ClpXP and other proteases return the high levels of several SOS proteins to homeostasis. When all SOS genes are constitutively expressed and many SOS proteins are stabilized by the removal of ClpXP, microscopic analysis shows that cells filament, produce mini-cells and have branching protrusions along their length. The only SOS gene required (of 19 tested) for the cell length phenotype is recN. ClpXP or recN4174 (A552S, A553V), a mutant not recognized by ClpXP, produce filamentous cells with nucleoid partitioning defects. It is hypothesized that when produced at high levels during the SOS response, RecN interferes with nucleoid partitioning and Z-Ring function by holding together sections of the nucleoid, or sister nucleoids, providing another way to inhibit cell division. RecN is a member of the Structural Maintenance of Chromosome (SMC) class of proteins. It can hold pieces of DNA together and is important for doublestrand break repair (DSBR). RecN is degraded by ClpXP. Overexpression of recN + in the absence of

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.

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