Genetic characteristics of new recA mutants of Escherichia coli K-12

Abstract

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...

Design and Evaluation of a Tryptophanless RecA Protein with Wild Type Activity

“…In addition to a putative role in interfilament contacts, the C-terminal domain (Trp290 and Trp308 in particular) may play a role in dsDNA binding (34,49). Mutations near the Trp positions in the C-terminal domain produce deleterious effects on both competition between RecA and SSB for ssDNA and secondary DNA binding by the nucleoprotein filament (34,50,51).…”

Biochemical basis of hyper‐recombinogenic activity of Pseudomonas aeruginosa RecA protein in Escherichia coli cells

“…β‐Galactosidase assay for spontaneous SOS‐induced gene expression was tested in strain GY7109: ΔrecA306 sfiA :: lacZ (R. Devoret's collection) carrying plasmids pUC19‐recA1.1 or pAK610 or pPA189.1. The NAL‐inducible variant of this assay was carried out as described previously (Alexseyev et al ., 1996).…”

A recombinational defect in the C‐terminal domain of Escherichia coli RecA2278‐5 protein is compensated by protein binding to ATP

“…On the other hand, the SOS response was only moderately defective at high temperature and the defect in UV repair was practically absent (Alexseyev et al, 1996). Being defective in recombination but not in DNA repair, recA2278-5 has a very unusual phenotype which makes it an interesting subject for biochemical examination.…”

“…A double mutation recA2278-5, featuring two amino acid substitutions, G-278→T and V-275→F in ␣-helix H of the RecA2278-5 protein, behaved almost as wild type at 32ЊC. However, at 42ЊC it was strongly deficient in conjugational recombination but only moderately affected in terms of the SOS response and the repair of UV-damaged DNA (Alexseyev et al, 1996).…”

“…To investigate the role of the C-terminal domain in various activities of RecA protein we constructed several functionally thermosensitive mutations in the ␣-helix H and in its close vicinity (Alexseyev et al, 1996). The rationale was to produce a conformational change in the C-terminal domain structure at a restrictive temperature.…”