Understanding radiation resistance using resistance‐promoting RecA mutants

Abstract

Multiple bacterial species exist that can survive the extensive DNA damage caused by ionizing radiation, yet the exact mechanisms of this resistance are not entirely understood. Using a directed evolution protocol, our lab has generated several strains of E. coli that are extremely radiation‐resistant. The genomes of multiple isolates from the evolved populations have been sequenced to identify mutations responsible for this phenotype. Two alleles were identified which encode mutations in RecA (A289S and D276A), a central protein in DNA repair. Inserting these alleles into wildtype E. coli resulted in small increases in survival after exposure to radiation. Thus, understanding the altered activity of these mutants can provide insight into DNA repair mechanisms and how an organism can survive ionizing radiation.RecA D276A displays an increased ability to bind to DNA coated with the single stranded DNA binding protein (SSB). This suggests that faster activation of RecA may aid in cell survival after DNA damage. Conversely, the RecA A289S protein has a diminished capability to bind to SSB‐coated DNA, and thus, the RecF, RecO, and RecR loading proteins may have a greater role in facilitating RecA binding to SSB‐coated DNA with this mutant. Also, RecA A289S filaments disassemble faster when exposed to the inhibitory RecX protein. This data suggests that tighter regulation of RecA A289S activity may produce a filament form that enhances certain types of RecA‐mediated recombination events. This work was supported by a grant from the National Institutes of Health, GM32335.