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

Genetics. 2020 Oct;216(2):381-393. doi: 10.1534/genetics.120.303476. Epub 2020 Aug 14.

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 a member of the Structural Maintenance of Chromosomes 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 ∆recN (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 ∆recN 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 (sensitive), 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 vivorecA4190 (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.

Keywords: DNA repair; SOS response; bacteria; homologous recombination.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • DNA Restriction Enzymes / genetics*
  • DNA Restriction Enzymes / metabolism
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Epistasis, Genetic*
  • Escherichia coli
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Mutation
  • Phenotype
  • Radiation Tolerance / genetics
  • Rec A Recombinases / genetics*
  • Rec A Recombinases / metabolism
  • Recombination, Genetic
  • Ultraviolet Rays

Substances

  • Bacterial Proteins
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • recA protein, E coli
  • Rec A Recombinases
  • DNA Restriction Enzymes
  • RecN protein, Bacteria