Location of functional regions of the Escherichia coli RecA protein by DNA sequence analysis of RecA protease-constitutive mutants

Abstract

In previous work (E. S. Tessman and P. K. Peterson, J. Bacteriol. 163:677-687 and 688-695, 1985), we isolated many novel protease-constitutive (Prtc) recA mutants, i.e., mutants in which the RecA protein was always in the protease state without the usual need for DNA damage to activate it. Most P&tC mutants were recombinase positive and were designated PrtC Rec+; only a few Prtc mutants were recombinase negative, and those were designated Prtc Rec-. We report changes in DNA sequence of the recA gene for several of these mutants. The mutational changes clustered at three regions on the linear RecA polypeptide. Region 1 includes amino acid residues 25 through 39, region 2 includes amino acid residues 157 through 184, and region 3 includes amino acid residues 298 through 301. The in vivo response of these Prtc mutants to different effectors suggests that the RecA effector-binding sites have been altered. In particular we propose that the mutations may define single-stranded DNA-and nucleoside triphosphate-binding domains of RecA, that polypeptide regions 1 and 3 comprise part of the single-stranded DNA-binding domain, and that polypeptide regions 2 and 3 comprise part of the nucleoside triphosphate-binding domain. The overlapping of single-stranded DNA-and nucleoside triphosphate-binding domains in region 3 can explain previously known complex allosteric effects. Each of four Prtc Rec-mutants sequenced was found to contain a single amino acid change, showing that the change of just one amino acid can affect both the protease and recombinase activities and indicating that the functional domains for these two activities of RecA overlap. A recA promoter-down mutation was isolated by its ability to suppress the RecA protease activity of one of our strong PrtC mutants.

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity

“…Mutations at residues Ser-117, Arg-243, and Gly-229 differentially affect this function (14,34). In addition, changes at Cys-116, Gly-157, and Arg-169 give coprotease-constitutive phenotypes (46,47).…”

“…However, evidence for an overlapping of these domains has also been presented elsewhere (7,46). To obtain further insights into the regulatory function of the RecA protein in both pathways, we created new recA mutants that had mutations in the vicinity of the recA430 mutation (Gly-204-*Ser) on the secondary structure of the protein.…”

Molecular cloning of the recA analog from the marine fish pathogen Vibrio anguillarum 775

“…If this interpretation is correct, carboxy-terminal portions of the RecA-like protein are important for DNA repair and recombination, but mutations in this region that nearly abolished recombinase activity resulted in a more subtle reduction in proteaselike activity. Singleamino-acid substitution mutations that affect both the recombinase and protease activities of E. coli RecA have been reported previously (12,45,47). In the case of mutations near the RecA carboxy terminus, dual effects on recombinase and protease activities were attributed to a defect in the single-stranded DNA-and nucleotide triphosphate-binding activities (47) that are required for recombination and activation of RecA.…”

“…Singleamino-acid substitution mutations that affect both the recombinase and protease activities of E. coli RecA have been reported previously (12,45,47). In the case of mutations near the RecA carboxy terminus, dual effects on recombinase and protease activities were attributed to a defect in the single-stranded DNA-and nucleotide triphosphate-binding activities (47) that are required for recombination and activation of RecA. Thus, it is possible that the carboxyterminal sequences of V. anguillarum RecA-like protein form a part of the single-stranded DNA-and/or nucleotide triphosphate-binding domain, such that mutations in this region affect both recombinase and protease activities.…”

Isolation and characterization of the Rickettsia prowazekii recA gene

“…E. coli Tyr-264, identified by Knight and McEntee (16) as the residue responsible for ATP binding, is also present in the rickettsial sequence (Tyr-264). In addition, E. coli residues associated with protease activity (Ser-25, Glu-158, Glu-38, Gly-157, Ile-128, Lys-177, and Arg-169) and homologous recombination (Gly-160, Leu-51, Arg-60, Arg-169, Ile-225, and Gly-301) are all conserved in rickettsial RecA (14,20,32). Finally, the rickettsial protein has retained the highly conserved proline residues observed by Tolmasky et al (29) …”