Filament Formation on Double‐Stranded DNA Mediates E38K RecA's Enhanced Activity in the Absence of DNA Damage

Abstract

RecA is a DNA recombinase implicated in DNA damage repair. The wild‐type version forms a filament on single‐stranded DNA (which is present after DNA damage), and this filament activates steps in the bacterial SOS response. Such activity includes stimulating the auto‐catalytic cleavage of the SOS gene repressor LexA and a subunit of the translesion DNA Polymerase V, UmuD. The E38K mutant variant of RecA shows an enhanced ability to perform these functions in the absence of DNA damage, and its expression produces a constitutive SOS response in vivo. Using ATPase, cleavage, and pH shift bioassays, I found that the constitutive SOS repsonse is mediated by E38K RecA's ability to form a filament on double‐stranded DNA (dsDNA). In the presence of dsDNA, E38K RecA hydrolyzed ATP at a superior rate compared to wild‐type RecA. Incubating LexA and UmuD in the presence of E38K RecA and dsDNA resulted in cleavage, while the same experiment with wild‐type RecA did not. Finally, by manipulating the pH of the in vitro solution to allow wild‐type RecA to form a filament on dsDNA, then reverting the pH back to physiological conditions (the filament persists), LexA cleavage was promoted. Overall, the enhanced function of E38K RecA in the absence of DNA damage is explained by its ability to form a filament on dsDNA. In addition, dsDNA is completely competent as a cofactor for RecA‐mediated LexA and UmuD cleavage.