Analysis of Escherichia coli RecA Interactions with LexA, λ CI, and UmuD by Site-Directed Mutagenesis of recA

Abstract

An early event in the induction of the SOS system of Escherichia coli is RecA-mediated cleavage of the LexA repressor. RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA. How complex formation leads to cleavage is not known. As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein. It was previously proposed that LexA and other cleavable substrates, such as phage CI repressor and E. coli UmuD, bind to a cleft located between two RecA monomers in the crystal structure. To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA. Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, CI, and UmuD. One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD. Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD. By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA. Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.

Several pathways of hydrogen peroxide action that damage the E. coli genome

“…Once activated, RecA interacts with LexA protein, the repressor of the SOS genes (Wagner et al, 1999). This interaction triggers the autocatalytic cleavage of LexA and consequent destruction of its ability to function as a repressor, which results in the derepression of SOS genes (Mustard and Little, 2000;Fernandez De Henestrosa et al, 2000). By using DNA microarray techniques Courcelle et al (2001) have shown that in E. coli the expression of 43 genes is controlled by LexA.…”

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

“…These proteins undergo autoproteolysis in vivo in a manner dependent on RecA, a protein involved in homologous recombination. The biological function of autocleavage is to inactivate these proteins (Mustard and Little 2000).…”

Mutagenic lesion bypass and two functionally different RecA proteins in Deinococcus deserti

“…E. coli RecA activity is autoregulated by its C-terminus; deletion of the last 17 residues enhances most RecA activities (Cox, 2007a,b). For the region of E. coli RecA that corresponds to residues 240-255 of D. deserti RecAC, analysis of point mutations and structural data have established that residues in this region are implicated in coprotease substrate binding and cleavage (Dutreix et al, 1989;Mustard and Little, 2000;McGrew and Knight, 2003;VanLoock et al, 2003).…”

“…Interestingly, one of the regions that are different in RecAC and RecAP, i.e. residues 240-255 (RecAC numbering), corresponds to a region in E. coli RecA that has been implicated in coprotease substrate binding and cleavage (Dutreix et al, 1989;Mustard and Little, 2000;McGrew and Knight, 2003;VanLoock et al, 2003). Therefore, the difference in this region of the RecA proteins might be an explanation for the inability of RecAP to induce TLS.…”