A Possible Role of the C-terminal Domain of the RecA Protein

Abstract

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270 -328 (from the N terminus). Our model building pointed out the possibility that this domain is a part of "gateway" through which double-stranded DNA finds a path for direct contact with single-stranded DNA within a presynaptic RecA filament in the search for homology. To test this possible function of the domain, we made mutant RecA proteins by site-directed single (or double, in one case) replacement of 2 conserved basic amino acid residues and 5 among 9 nonconserved basic amino acid residues in the domain. Replacement of either of the 2 conserved amino acid residues caused deficiencies in repair of UV-damaged DNA, an in vivo function of RecA protein, whereas the replacement of most (except one) of the tested nonconserved ones gave little or no effect. Purified mutant RecA proteins showed no (or only slight) deficiencies in the formation of presynaptic filaments as assessed by various assays. However, presynaptic filaments of both proteins that had replacement of a conserved amino acid residue had significant defects in binding to and pairing with duplex DNA (secondary binding). These results are consistent with our model that the conserved amino acid residues in the C-terminal domain have a direct role in doublestranded DNA binding and that they constitute a part of a gateway for homologous recognition.RecA protein is a key enzyme in homologous genetic recombination and recombinational repair of damaged DNA. RecA protein, which consists of 352 amino acid residues, promotes the formation of heteroduplex joints between single-stranded and double-stranded DNA by homologous pairing and strand exchange in vitro (1, 2). In homologous pairing, RecA protein first binds to single-stranded DNA in the presence of ATP ("primary binding"; Ref.3) and forms helical nucleoprotein filaments called "presynaptic filaments," in which singlestranded DNA is extended to a length 1.5 times longer than the B form DNA (4 -7). Then, presynaptic filaments bind to doublestranded DNA ("secondary binding") and form a three-component complex including single-stranded DNA, double-stranded DNA, and RecA protein (and ATP) without prior homologous alignment. The search for homology between single-stranded and double-stranded DNA occurs in this three-component complex (3,8,9).We had shown that a chimeric RecA protein of Escherichia coli and Pseudomonas aeruginosa, RecAc38, is defective in homologous pairing of single-stranded DNA with double-

Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA

“…Remarkably, both the C-terminal domain of RecA (16,17) and the N-terminal domain of Rad51 (18) have been shown to bind DNA. As with the helicases, these domains associated with the nucleotide-binding core have no structural homology (18,37).…”

“…The homologous core structure has also been found in the F1-ATPase (11) and in several helicases (12)(13)(14)(15), suggesting that all of these proteins have diverged from a common ancestor. Although there is no apparent homology between the N-terminal domain of Rad51 and the C-terminal domain of RecA, it has been reported that the C-terminal domain of RecA binds double-stranded DNA (dsDNA) (16,17) and that the N-terminal domain of hRad51 binds both single-stranded DNA (ssDNA) and dsDNA (18).…”

Origins and evolution of the recA / RAD51 gene family: Evidence for ancient gene duplication and endosymbiotic gene transfer

“…In contrast, eubacterial and plant RecA proteins contain a conserved Cterminal domain that is absent in other members. The RecA C-terminal domains also bind to double-stranded DNA (23) and are similar in function to, but distinct in sequence from, the N-terminal domain of RAD51 and DMC1 (24,25). Plant RecA proteins also contain a Ϸ70-aa N-terminal region that is different from the RADA͞RAD51 N-terminal regions.…”

Biochemical Characterization of the Human RAD51 Protein

“…In addition, hRAD51 protein has an extended amino terminus and truncated carboxyl terminus relative to the bacterial RecA (28,29). The amino terminus of RecA forms extensive contacts between neighboring protomers within a filament and is critical for self-association (30), while the carboxyl terminus of RecA exists as a distinct domain that appears to modulate DNA binding (31,32). Interestingly, a study that combined NMR and mutagenesis indicated that the extended amino terminus of hRAD51 may functionally replace the carboxyl-terminal domain of RecA (33).…”