Differential Requirements of Two recA Mutants for Constitutive SOS Expression in Escherichia coli K-12

Background Repairing DNA damage begins with its detection and is often followed by elicitation of a cellular response. In E. coli, RecA polymerizes on ssDNA produced after DNA damage and induces the SOS Response. The RecA-DNA filament is an allosteric effector of LexA auto-proteolysis. LexA is the repressor of the SOS Response. Not all RecA-DNA filaments, however, lead to an SOS Response. Certain recA mutants express the SOS Response (recAC) in the absence of external DNA damage in log phase cells. Methodology/Principal Findings Genetic analysis of two recAC mutants was used to determine the mechanism of constitutive SOS (SOSC) expression in a population of log phase cells using fluorescence of single cells carrying an SOS reporter system (sulAp-gfp). SOSC expression in recA4142 mutants was dependent on its initial level of transcription, recBCD, recFOR, recX, dinI, xthA and the type of medium in which the cells were grown. SOSC expression in recA730 mutants was affected by none of the mutations or conditions tested above. Conclusions/Significance It is concluded that not all recAC alleles cause SOSC expression by the same mechanism. It is hypothesized that RecA4142 is loaded on to a double-strand end of DNA and that the RecA filament is stabilized by the presence of DinI and destabilized by RecX. RecFOR regulate the activity of RecX to destabilize the RecA filament. RecA730 causes SOSC expression by binding to ssDNA in a mechanism yet to be determined

RecA4142 Causes SOS Constitutive Expression by Loading onto Reversed Replication Forks in Escherichia coli K-12 ▿

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 (5′→3′ exonuclease), xonA (3′→5′ 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

This figure shows the distributions of cells with different levels of constitutive SOS expression (detected as GFP fluorescence) expressed as the percentage of cells in the population.

<p>The graphs truncate the percentage of cells at 25%. The strains are in order from top of the graph to the bottom with the relevant part of the genotype in parentheses. Unless otherwise indicated, all strains were grown in minimal medium at 37°C with aeration. The strains are: SS1408 (<i>lexA51::Tn5</i>), SS4629 (<i>recA730</i>), SS4976 (<i>recAo1403 recA4142</i>), SS6013 (<i>recA4142</i>), SS6088 (<i>recAo1403 recA⁺</i>) and SS996 (<i>recA</i>⁺).</p

Same as for Figure 2.

<p>SS4976 (<i>recAo1403 recA4142</i>), SS5312 (<i>recAo1403 recA4142 del(recX)</i>) SS6023 (<i>recAo1403 recA4142 del(recBCD)::cat</i>), SS6048 (<i>recAo1403 recA4142 del(recBCD)::cat del(recX)</i>), SS4696 (<i>recAo1403 recA4142 recF4115</i>), SS5394 (<i>recAo1403 recA4142 recF4115 del(recX)</i>).</p

Same as for Figure 2.

<p>All grown in rich medium: SS996 (<i>recA</i>⁺), SS6080 (<i>del(recX)</i>), SS6013 (<i>recA4142</i>), SS6019 (<i>recA4142 del(recX)</i>), SS4976 (<i>recAo1403 recA4142</i>), SS5312 (<i>recAo1403 recA4142 del(recX)</i>).</p

Same as for Figure 2.

<p>SS4629 (<i>recA730</i>), SS6044 (<i>recA730 del(recBCD)::cat</i>), SS4645 (<i>recA730 recF4115</i>), SS5316 (<i>recA730 del(dinI)</i>), SS4976 (<i>recAo1403 recA4142</i>), SS6023 (<i>recAo1403 recA4142 del(recBCD)::cat</i>), SS4696 (<i>recAo1403 recA4142 recF4115</i>), SS5315 (<i>recAo1403 recA4142 del(dinI)</i>).</p

Summary of phenotypic analysis of <i>recA</i> mutants used in this study.

a<p>ND is Not Determined because the cells are already fully induced for SOS expression.</p

Strains used in this work.

a<p>JC13509 has the following genotype: <i>sulB103 lacMS286 φ 80dIIlacBK1 argE3 hi-4 thi-1 xyl-5 mtl-1 rpsL31 tsx</i>. The <i>lacMS286φ80dIIlacBK1</i> code for two partial non-overlapping deletions of the <i>lac</i> operon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004100#pone.0004100-Konrad1" target="_blank">[73]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004100#pone.0004100-Zieg1" target="_blank">[74]</a>.</p>b<p>Select for Kan^R and then screen for other marker phenotypically or by PCR.</p>c<p>Select for Tet^R and then screen for other marker phenotypically or by PCR.</p>d<p>Select for Cat^R and then screen for other marker phenotypically or by PCR.</p>e<p>Select for Amp^R.</p>f<p>Select for AlaS⁺.</p>g<p>This deletion allele was created by first transducing the kan resistant derivative from the Kieo collection into the strain as indicated in the reference column. pLH29, carrying the <i>flp</i> gene, was then introduced and Kan sensitive derivatives were screened (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004100#pone.0004100-Huang1" target="_blank">[75]</a>.</p>h<p><i>recX::cat</i> was amplified with prSJS748,749 using pACYC184 (New England Biolabs) as a template. <i>recX::cat</i> was transferred to the chromosome using the <i>exo-bet</i> method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004100#pone.0004100-Datsenko1" target="_blank">[76]</a> next to the <i>recA</i> allele indicated. This original combination of mutants were named and saved as the strain indicated as the donor in this cross.</p>i<p>These <i>recAo</i> or <i>recA</i> mutations were first constructed on a plasmid as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004100#s4" target="_blank">Materials and Methods</a>. They were then transferred to the chromosome using the method of Datsenko and Wanner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004100#pone.0004100-Datsenko1" target="_blank">[76]</a> using a strain that was <i>lexA3 malE::Tn10</i> in a JC13509 background with pKD46 encoding <i>exo</i> and <i>bet</i>. This original combination of mutants were named and saved as the strain indicated as the donor in this cross.</p>j<p>Full notation for <i>ygaD</i> mutation is <i>ygaD1::kan</i> .Full notation for <i>recX</i> mutation is <i>del(recX)4166::cat</i>. Full notation for <i>recBCD</i> mutation is <i>del(recBCD)::cat</i>. Full notation for Ω<i>gfp</i> mutation is <i>Δattλ::sulApΩgfp-mut2</i>.</p

Oligonucleotide primers used in this work.

<p>Oligonucleotide primers used in this work.</p

Same as for Figure 2.

<p>SS6013 (<i>recA4142</i>) minimal, SS6013 (<i>recA4142</i>) rich, SS4976 (<i>recAo1403 recA4142</i>) minimal, SS4976 (<i>recAo1403 recA4142</i>) rich.</p