SummaryThe replacement of Escherichia coli recA gene (recA Ec protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH 7.5. We hypothesized that the high affinity of RecA Pa protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.

In vivo, the RecA protein of Escherichia coli plays a critical role in such cellular functions as homologous recombination, repair of damaged DNA, and the SOS response (see references 10, 12, and 21 for reviews). To mediate necessary biochemical reactions, the RecA protein must be in an active form that combines three ingredients (RecA, ATP, and singlestranded DNA) in a helical RecA nucleoprotein filament (17).The current view of the physical structure of the filament is based on X-ray analysis of RecA crystals (23). According to that analysis, the RecA monomer is composed of three domains, one major domain and two subdomains located at its amino and carboxyl termini. Both the major domain and the N-terminal domain participate in the formation of RecA multimers, the structure of which has been suggested to resemble closely that of the RecA-DNA filament (23). The C-terminal domain protrudes from the multimer. While it does not participate in an intrafilament interaction, it does take part in an interfilament interaction. The latter has been recently discussed as a possible step in the recombination exchange reaction (13).The C-terminal domain consists of three ␣ helices (H, I, and J) that are situated orthogonally and two ␤ strands (␤9 and ␤10) located between the H and I helices (23). Epitope mapping of anti-RecA protein monoclonal immunoglobulins G, ARM191 and ARM193, which were suggested to affect, respectively, the site for interaction of RecA monomers within the RecA filament and the site for interaction between RecA and double-stranded DNA, showed that the antibodies cover the region of the C-terminal domain including at least ␤-strand 10, as well as helices I and J (7). The functional role of ␣-helix H and adjacent ␤-strand 9 remains unclear. We used sitedirected mutagenesis to elucidate the possible functions of these two adjacent structures.The other reason for our mutagenesis study was to search for functionally thermosensitive (FT) mutations. To date, only two FT mutations, recA44 (8) and recA200 (14), have been described. The former has been localized. We sequenced the recA2283 allele, which was responsible for the RecA200 FT phenotype. The analysis of both recA44 and recA2283 mutations helped us to find a strategy for searching for other thermosensitive mutations in this and other regions. The rationale was to design amino acid substitutions which must be functionally related to the original residues.Besides the C-terminal domain, two regions of the major domain, ␤-strand 6 and ␣-helix F, were involved in the construction of new mutations. The choice of the former region was dictated by an expectation, based on the properties of known recA mutations from this area, that it should be possible to reveal the site responsible for regulation of interaction between the RecA protein and the LexA repressor (see reference 19 for a review). The ␣-helix F region belongs to the type of polypeptide motifs predicted by Churchill and Travers (3) as that which might recognize structural features of DNA.In this repor...

SummaryRecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the ␣-helix H of the C-terminal subdomain of the protein. recA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42ЊC) in the absence of ATP resulted in inhibition at 42ЊC of many activities related to homologous recombination including ss-and dsDNA binding, high-affinity binding for ATP, ss-or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5 M) was shown to be another factor that can stabilize the RecAmut protein at 42ЊC, judging by restoration of its DNA-free ATPase activity. The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation.The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecAmut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.