Functional overlap between RecA and MgsA (RarA) in the rescue of stalled replication forks in Escherichia coli

Abstract

Escherichia coli RecA protein plays a role in DNA homologous recombination, recombination repair, and the rescue of stalled or collapsed replication forks. The mgsA ( rarA ) gene encodes a highly conserved DNAdependent ATPase, whose yeast orthologue, MGS1 , plays a role in maintaining genomic stability. In this study, we show a functional relationship between mgsA and recA during DNA replication. The mgsA recA double mutant grows more slowly and has lower viability than a recA single mutant, but they are equally sensitive to UV-induced DNA damage. Mutations in mgsA and recA cause lethality in DNA polymerase I deficient cells, and suppress the temperature-dependent growth defect of dnaE486 (Pol III α α α α -catalytic subunit). Moreover, recAS25P , a novel recA allele identified in this work, does not complement the slow growth of ∆ ∆ ∆ ∆ mgsA ∆ ∆ ∆ ∆ recA cells or the lethality of polA12 ∆ ∆ ∆ ∆ recA , but is proficient in DNA repair, homologous recombination, SOS mutagenesis and SOS induction. These results suggest that RecA and MgsA are functionally redundant in rescuing stalled replication forks, and that the DNA repair and homologous recombination functions of RecA are separated from its function to maintain progression of replication fork.

Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

“…Our results showed that T. kodakaraensis cells are originally more sensitive to UV, as compared to other archaeal species, such as S. solfataricus (Wood et al, 1997), H. volcani (McCready, 1996, and Halobacterium sp. NRC-1 (Baliga et al, 2004), and are also more sensitive than typical model organisms, such as E. coli (Shibata et al, 2005), and Saccharomyces cerevisiae (Hishida et al, 2002). The higher sensitivity of T. kodakaraensis to UV can be rationalized by considering that this organism was isolated from the bottom of the sea, an environment far from UV sources (Morikawa et al, 1994).…”

RecA Protein Recruits Structural Maintenance of Chromosomes (SMC)-like RecN Protein to DNA Double-strand Breaks

“…Isolation of recA Mutant-A library of recA mutants was generated by carrying out PCR-mediated random mutagenesis, as described previously (36). The resultant recA mutant clones were transformed into a recA strain.…”

“…Mitomycin C (2.5 mg/ml) was dissolved in 10 mM Tris-HCl (pH 8.5) buffer. Sensitivity to UV damage was measured as described previously (36). To measure sensitivity to MMC, cultures were grown in LB broth to an A 650 of ϳ0.4, serially diluted, spotted onto LB medium containing the indicated concentration of MMC, and incubated at 37°C.…”

“…SOS Induction-SOS induction was assessed by measuring the degradation of LexA as described previously (36). Log phase cultures were treated with MMC (0.5 g/ml), and aliquots were taken at multiple time points for immunoblot analysis with anti-LexA (BioAcademia), anti-RecA (BioAcademia), or antiRecN (26).…”

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

“…The kan gene flanked by flippase recognition target (FRT) was then eliminated using the flippase helper plasmid pCP20 (Datsenko and Wanner, 2000). The temperaturesensitive dnaE486 mutation of TS1502 (Shibata et al, 2005) was cotransduced with the tetracycline resistance gene of Tn10 into recipient strains at 30°C. Temperaturesensitive colonies were selected among tetracyclineresistant transductants, yielding MK7146 from MK7136 and MK7149 from MK7140.…”