RecA4142 Causes SOS Constitutive Expression by Loading onto Reversed Replication Forks inEscherichia coliK-12

Abstract

Escherichia coli initiates the SOS response when single-stranded DNA (ssDNA) produced by DNA damage is bound by RecA and forms a RecA-DNA filament. recA SOS constitutive [recA(Con)] mutants induce the SOS response in the absence of DNA damage. It has been proposed that recA(Con) mutants bind to ssDNA at replication forks, although the specific mechanism is unknown. Previously, it had been shown that recA4142(F217Y), a novel recA(Con) mutant, was dependent on RecBCD for its high SOS constitutive [SOS(Con)] expression. This was presumably because RecA4142 was loaded at a double-strand end (DSE) of DNA. Herein, it is shown that recA4142 SOS(Con) expression is additionally dependent on ruvAB (replication fork reversal [RFR] activity only) and recJ (533 exonuclease), xonA (335 exonuclease) and partially dependent on recQ (helicase). Lastly, sbcCD mutations (Mre11/Rad50 homolog) in recA4142 strains caused full SOS(Con) expression in an ruvAB-, recBCD-, recJ-, and xonA-independent manner. It is hypothesized that RuvAB catalyzes RFR, RecJ and XonA blunt the DSE (created by the RFR), and then RecBCD loads RecA4142 onto this end to produce SOS(Con) expression. In sbcCD mutants, RecA4142 can bind other DNA substrates by itself that are normally degraded by the SbcCD nuclease.The SOS response is a coordinated response of Escherichia coli at the level of transcription to DNA damage (10,18,26). RecA initiates this response by binding to single-stranded DNA (ssDNA) produced by DNA damage and serving as an allosteric effector for auto-proteolysis of the LexA transcriptional repressor. The transcription of at least 40 genes is increased during the SOS response (16). Some of the induced genes include recA, ruvAB, dinI, and recX. The RecA protein also plays a central role in recombinational repair and homologous recombination (8,11,22). It participates in all three processes through its ability to polymerize on ssDNA to create a RecA-DNA filament.Several proteins are known to either help RecA load onto different DNA substrates or regulate the stability of the RecA-DNA filament (reviewed in reference 12). One of the loading complexes is RecBCD. This complex has the ability to load onto a double-strand end (DSE) of DNA. It processes the DNA using helicase, exonuclease, and Chi recognition activities to produce a region of ssDNA with a 3Ј end. RecBCD then loads RecA onto this ssDNA, creating a RecA-DNA filament (reviewed in reference 14). Another complex, RecFOR, loads RecA onto ssDNA coated with single-stranded DNA binding protein (SSB) at a gapped DNA substrate (36, 38). After production, DinI stabilizes and RecX destabilizes RecA filaments (29, 37). RecFOR antagonizes the destabilization activity of RecX both in vivo and in vitro (28, 30).There are certain mutants of recA that turn on SOS expression in the absence of external DNA damage. These are called recA constitutive [recA(Con)] mutants (reviewed in reference 33). While the specific mechanism of how this type of mutant induces SOS is presently unknown, it is thought that it o...

Factors Limiting SOS Expression in Log-Phase Cells of Escherichia coli

“…It was found that SOS expression in recA4142 (F217Y) mutants was dependent on its initial level of transcription, recBCD, ruvAB, recJ, and xonA. The data are consistent with the model that the loading of RecA4142 occurs at reversed replication forks (28). Furthermore, it was found that recX and xthA repressed the level of SOS constitutive expression in the recA4142 mutant (29).…”

“…Constitutive SOS expression in the recA4142 (F217Y) mutant is dependent upon ruvAB (28,29). Since radA is partially redundant with ruvAB for UV survival and recombination (1), it is possible that radA may also be required for SOS expression in a recA4142 mutant and that the deletion of radA should lower SOS expression.…”

“…Recent studies have analyzed recA mutants constitutive for SOS in the absence of external DNA damage (i.e., recA4142 mutants) and characterized the requirements for this SOS expression (28,29). It was found that SOS expression in recA4142 (F217Y) mutants was dependent on its initial level of transcription, recBCD, ruvAB, recJ, and xonA.…”

Specificity in suppression of SOS expression by recA4162 and uvrD303

“…Several of these types of mutants have been described (reviewed in [36]). It has been shown for two of these mutants, recA4142 (F217Y) and recA730 (E38K), that while they both cause SOS constitutive (SOS C ) expression, they do so through different mechanisms [37–39]. SOS C expression in recA4142 mutants depends on several genes: recBCD, ruvAB, recJ and sbcB [37].…”

“…It has been shown for two of these mutants, recA4142 (F217Y) and recA730 (E38K), that while they both cause SOS constitutive (SOS C ) expression, they do so through different mechanisms [37–39]. SOS C expression in recA4142 mutants depends on several genes: recBCD, ruvAB, recJ and sbcB [37]. It was proposed that RecBCD loads RecA4142 onto the ends of a replication fork that has been reversed by RuvAB and tailored by RecJ and SbcB.…”