Two RecA Protein Types That Mediate Different Modes of Hyperrecombination

Abstract

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.The bacterial RecA protein is multifunctional (11,17,25). In Escherichia coli, the RecA protein is first a recombinase, promoting homologous recombination and recombinational DNA repair. Second, RecA filaments formed on DNA facilitate the autocatalytic cleavage of the LexA protein to allow induction of the SOS response. This activity is referred to as the RecA coprotease function. Finally, RecA is directly involved in the activation of DNA polymerase V, the polymerase responsible for SOS mutagenesis (11,17,25). The present study focused on RecA function in recombination and recombinational DNA repair.The key steps in homologous recombination promoted by a RecA nucleoprotein filament are (i) formation of the presynaptic filament (a ternary complex including RecA, ATP, and single-stranded DNA [ssDNA]) in the presence of magnesium ion, (ii) pairing of the presynaptic filament with homologous double-stranded DNA (dsDNA), and (iii) DNA strand exchange between ssDNA and dsDNA that results in a new heteroduplex dsDNA and a displaced ssDNA (11,15,17,25,37). To promote these reactions, RecA has two main activities, the DNA-dependent ATPase, activated with both ssDNA and dsDNA, and DNA strand transferase. The latter activity can be broken down into several characteristics commonly used for the comparison of different RecA proteins. These include RecA protein binding to ssDNA to form a presynaptic complex (PC), RecA protein-mediated displacement of SSB protein from ssDNA, the stability of PCs (for ex...

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering

“…E. coli RecA was purified from the strain JC10289 with the relevant genotype recA thr‐1 leu‐6 Str r [32] carrying the wild type recA gene under its own promoter within the plasmid pUC19‐recA1.1 [33]. The cells were grown at 37 °C in 2 l of Luria–Bertani medium in the presence of 100 μg/ml ampicillin to an OD 595 of 0.6 and the protein expression was induced for 3 h by the addition of nalidixic acid (50 μg/ml).…”

Modulating cellular recombination potential through alterations in RecA structure and regulation

“…Measurement of FRE to assess elevated or depressed levels of recombination in vivo has been developed over the past 15 years (Namsaraev et al ., 1998; Bakhlanova et al ., 2001; Chervyakova et al ., 2001; Lanzov, 2002; Baitin et al ., 2003; 2006; 2008; Lanzov et al ., 2003). The method has the advantage that it can detect essentially all types of genetic exchanges that might occur during conjugation (Baitin et al ., 2008). The method is somewhat more laborious than the convenient and robust method developed by Konrad (Konrad, 1977).…”

“…It is also not clear to what extent recombination potential reflects RecA protein structure itself, the modulation of RecA protein function by other factors or the activities of entirely different recombination proteins. Therefore, to directly address the affects of these variables on the in vivo function of RecA, we took advantage of a quantitative genetic analysis of the linkage of donor markers following bacterial conjugation by determining the frequency of recombination exchanges per DNA unit length (FRE) (Namsaraev et al ., 1998; Bakhlanova et al ., 2001; Chervyakova et al ., 2001; Lanzov, 2002; Baitin et al ., 2003; 2006; 2008; Lanzov et al ., 2003). These methods have shown that RecA from E. coli (EcRecA) has a relatively moderate recombinase activity in vivo (Namsaraev et al ., 1998; Lanzov et al ., 2003; Baitin et al ., 2006; 2008), although the activity is presumably optimal for this bacterium.…”

“…Hyper‐recombination arises as a result of the induction of the SOS response, either transiently by treating normal cells with a DNA damaging agent (Sassanfar and Roberts, 1990) or constitutively as observed with certain RecA mutant variants such as RecA E38K ( recA 730) (Lavery and Kowalczykowski, 1992). Hyper‐recombination can also be SOS‐independent, as observed in a set of RecA proteins including the Pseudomonas aeruginosa RecA (PaRecA) (Namsaraev et al ., 1998; Baitin et al ., 2003; 2006) and different E. coli / P. aeruginosa chimeric RecAX proteins (Bakhlanova et al ., 2001; Baitin et al ., 2006; 2008) expressed in E. coli .…”

Paired repeats in the structure of the bacterial genome and recombination activity in cells

“…The protein RecA of pathogenic Pseudomonas aeruginosa (RecAPa), for example, exhibits hyperrecombination increasing FRE by six to seven times, and the hybrid protein RecAX53, the central domain of which consists of 12 amino acids from RecAPa, while the rest consists of 340 amino acids from RecAEc, increases FRE by nine times (Bakhlanova et al, 2001). In contrast to RecAEc, the RecAPa and RecAX53 proteins have, however, an increased biochemical activity that influ ences the recombination rate (Baitin et al, 2008).…”