An Epistasis Analysis of recA and recN in Escherichia coli K-12

Abstract

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.

SMC protein RecN drives RecA filament translocation and remodelling for in vivo homology search

“…Consistent with reports in other bacterial systems 50,52 , we find that RecN is essential for successful DSB repair in Caulobacter. In absence of recN we record no detectable repair events, together with an increased sensitivity to DSB-inducing agents (Fig.…”

“…We now inquire into the molecular basis of the dynamics of the RecA filament during homology search and for holding break ends together during filament translocation. An essential and highly conserved member of the bacterial recombination pathway is the SMC-like protein, RecN 50,52 . However, it still remains unclear as how RecN influences RecA-mediated homologous recombination in vivo .…”

“…In addition, several accessory proteins are reported to modulate the association and turnover of RecA with ssDNA (19,20). Of these, RecN, a highly conserved repair-associated protein, has been shown to interact with RecA and is essential for DSB repair (20)(21)(22)(23). RecN belongs to the Structural Maintenance of Chromosome (SMC) family of proteins, that play central roles in chromosome dynamics (24).…”

SMC protein RecN drives RecA filament translocation for in vivo homology search

“…In addition, several accessory proteins are reported to modulate the association and turnover of RecA with ssDNA ( 42 , 43 ). Of these, RecN, a highly conserved repair-associated protein, has been shown to interact with RecA and is essential for DSB repair ( 43 – 47 ). RecN belongs to the Structural Maintenance of Chromosome (SMC) family of proteins that play central roles in chromosome dynamics ( 48 ).…”

The SMC-like RecN protein is at the crossroads of several genotoxic stress responses in Escherichia coli

“…Because RecN interacts with RecA ( Vickridge et al, 2017 ) and both are equivalently required to survive I-SCE 1 mediated DSB, they are generally associated in the same epistatic group ( Meddows et al, 2005 ). Recent data, however, suggests that RecA and RecN may also function in genetically distinct pathways important for DNA repair ( Klimova and Sandler, 2020 ).…”