Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions.

Abstract

The RadA/Sms protein is a RecA-related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double-strand break repair for YejH.