engleski

The 5’-3’exonuclease is essential for high constitutive SOS expression in recA730 mutants of Escherichia coli

The RecA protein is a central component of the recombination machinery which is required for double-strand break (DSB) repair, repair of single stranded gaps (SSG), production of genetic variation during conjugation and induction of the SOS response. In wild-type (wt) Escherichia coli strains, DSBs are processed into RecA filaments by the RecBCD pathway, whereas SSGs utilize the RecF recombination pathway [1]. In a recB1080 mutant, the components of the two recombination machineries act together to produce a RecA filament [2]. There are three enzymatic activities essential for the RecA filament formation: helicase, 5’-3’ exonuclease, and RecA loading onto single-stranded DNA [1]. We studied a specific recA mutant named recA730 (RecAE38K) which encodes a form of RecA protein that is able to suppresses recombination and DNA repair deficiency in cells where both mechanisms for RecA filament formation are inactivated by mutations in genes for mediator proteins involved in RecA loading [3]. This gain of function mutant also exhibits high constitutive SOS expression (cSOS) [4]. The SOS response involves the elevated expression of more than 50 genes with various functions that are induced in response to damage of chromosomal DNA [5]. By studying the genetic requirements for high cSOS expression in recA730 mutants, we found that three different genetic backgrounds (wt, recB1080 and recB null), have different genetic requirements. In wt background, the high cSOS expression of a recA730 mutant is partially dependent on RecBCD function, implying that RecBCD can moderately enhance the already excellent intrinsic abilities of the RecA730 enzyme. In recB1080 background, the high cSOS expression of the mutant is partially dependent on the helicase activity of the RecB1080CD enzyme and is strongly dependent on the RecJ nuclease. The cSOS expression of a recA730 mutant in a recB null background is dependent on the RecJ nuclease. These results emphasize the importance of the 5’-3’exonuclease for high cSOS expression in recA730 mutants. [1] Kowalczykowski, S.C. (2000) Initiation of genetic recombination and recombination-dependent replication. Trends Biochem. Sci. 25:156-165. [2] Ivančić-Baće, I. et al. (2003) RecFOR function is required for DNA repair and recombination in a RecA loading-deficient recB mutant of Escherichia coli. Genetics 163: 485-494. [3] Vlašić, I. et al. (2011) The recA730 dependent suppression of recombination deficiency in RecA loading mutants of Escherichia coli. Res. Microbiol. 162: 262-269. [4] Long, J. E. et al. (2008) Differential requirements of two recA mutants for constitutive SOS expression in Escherichia coli K-12. PLoS One 3:e4100. [5] Michel, B. (2005) After 30 years of study, the bacterial SOS response still surprises us. PLoS Biol. 3:e255.

constitutive SOS; Escherichia coli; recA730

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