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Effect of Δ<i>rnhA</i> and Δ<i>rnhB</i> on UV survival of <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains expressing pol V variants.

<p>10 µl of 10-fold serial dilutions of overnight cultures were spotted onto the surface of rectangular LB agar plates and exposed to 40 J/m² 254 nM UV-light (panels A and C) and 20 J/m² 254 nM UV-light (panels B and D). Both unirradiated (−) and UV-irradiated (+) plates were incubated overnight at 37°C. In each panel, UV survival is shown for the <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains either harboring pGB2 vector, or expressing pol V variants. The main observation of these experiments is that the UV-resistance of cells expressing <i>umuC</i>_Y11A increase dramatically in strains lacking <i>rnhB</i>, whereas survival of cells equipped with wild-type pol V, <i>umuC</i>_F10L, or <i>umuC</i>_Y11F is largely unaffected by the status of <i>rnhB</i>.</p

Mechanisms Employed by <em>Escherichia coli</em> to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V

<div><p><em>Escherichia coli</em> pol V (UmuD′₂C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs <em>in vitro.</em> Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A <em>in vitro</em>, strains expressing <em>umuC</em>_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by <em>umuC</em>_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV <em>in vivo</em>. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass <em>in vitro</em>. In a <em>recA730 lexA</em>(Def) Δ<em>umuDC</em> Δ<em>dinB</em> strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, <em>umuC</em>_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic Δ<em>rnhB</em> strain and ∼72% of wild-type levels in a Δ<em>rnhA</em> Δ<em>rnhB</em> double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the <em>E. coli</em> genome, but at the cost of higher levels of cellular mutagenesis.</p> </div

Quantitative UV survival and mutagenesis assays.

<p>A: Survival. Exponentially growing cells were exposed to various doses of UV-light and serial dilutions spread on LB plates containing spectinomycin. The number of viable colonies was determined after overnight incubation at 37°C. Error bars indicate the standard error of the mean. Consistent with the semi-quantitative UV-survival assay shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003030#pgen-1003030-g001" target="_blank">Figure 1</a>, UV-resistance of strains expressing Y11A_UmuC increased significantly in the Δ<i>rnhB</i> background, while there was no change in UV-survival of the strain harboring vector, pGB2, or expressing wild-type pol V in the <i>rnhB</i>^+/− strains. B: Mutagenesis. UV-induced mutagenesis was determined by exposing exponentially growing cells to 20 J/m² UV light. Cell viability was in the range of 85–90% survival for wild-type pol V and ∼60–70% for vector control, pGB2, and the Y11A mutant. The average number of His⁺ revertants per 10⁸ surviving cells ± standard error of the mean is indicated on the graph. The <i>rnhB</i>⁺ strains are indicated by navy-colored bars, while Δ<i>rnhB</i> strains are indicated by the gold-colored bars. As observed, the UmuC_Y11A-expressing cells exhibited an ∼9-fold increase in UV mutagenesis compared to the <i>rnhB</i>⁺ strain.</p

Effect of Δ<i>rnhA</i> and Δ<i>rnhB</i> on spontaneous mutagenesis in <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains expressing pol V variants.

<p>Spontaneous mutagenesis was measured by assaying reversion of the <i>hisG4</i> ochre allele (leading to <i>histidine</i> prototophy) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003030#s4" target="_blank"><i>Materials and Methods</i></a>. The average number of His⁺ revertants per plate ± standard error of the mean is indicated in the table. Since the extent of mutagenesis promoted by wild-type pol V differed in the various strains, we have expressed the level of mutagenesis promoted by the variants as a percentage of wild-type mutagenesis. As clearly observed, <i>umuC</i>_Y11A-dependent mutagenesis increased in the Δ<i>rnhB</i> strain and was further elevated in the Δ<i>rnhA</i> Δ<i>rnhB</i> double mutant. In contrast, <i>umuC</i>_F10L gave consistently low levels of mutagenesis in all strains, and <i>umuC</i>_Y11F higher than wild-type levels in all strains.</p

Role of NER in strains expressing pol V variants.

<p>10 µl of 10-fold serial dilutions of overnight cultures were spotted onto the surface of rectangular LB agar plates and exposed to 40 J/m² 254 nM UV-light (Panel A), or 1 J/m² 254 nM UV-light (Panels B and C). Both unirradiated (−) and UV-irradiated (+) plates were incubated overnight at 37°C. In each panel, UV survival is shown for the <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains either harboring pGB2 vector, or expressing pol V variants. Panel A (<i>uvr</i>⁺ strain) is reproduced from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003030#pgen-1003030-g001" target="_blank">Figure 1A</a> for direct comparison to the isogenic Δ<i>uvrA</i> (Panel B) and Δ<i>uvrC</i> (panel C) strains. The main observation of these experiments is that while the <i>uvr</i>⁻ strains are considerably more UV-sensitive than the isogenic <i>uvr</i>⁺ strain, the relative sensitivity of the strains expressing pol V variants changes in the <i>uvr</i>⁻ background, with UmuC_Y11A promoting an increase in UV-survival to a similar extent as wild-type pol V.</p

Model for the effect of RNase HII and NER proteins on UV sensitivity of strains proficient for ribonucleotide incorporation.

<p>Translesion replication catalyzed by Y11A mutant produces a TLS tract containing multiple ribonucleotides. NER excises UV-induced lesions and produces gaps on the template strand. RNase HII initiating removal of multiple rNMPs incorporated during TLS produces nicks on the daughter strand. The concerted action of both these repair pathways results in formation of persistent double strand breaks ultimately leading to cell death. Inactivation of either repair pathway selectively improves UV-resistance of cells expressing UmuC_Y11A.</p

<i>In vitro</i> translesion synthesis past a TT-CPD lesion catalyzed by mixtures of pol IV and pol V.

<p>Translesion DNA synthesis was performed using a circular DNA template with a running-start primer with its 3′ end located 5 bases before the 3′T of the CPD. Primer extension reactions catalyzed by pol IV (80 nM, lane 1 or 800 nM, lane 6), wild-type pol V (80 nM, lanes 2 and 7), pol V (UmuC_Y11A) (60 nM, lanes 4 and 9), or a combination of pol IV (80 nM, lane 3 and 5 or 800 nM, lane 8 and 10) with either wild-type (80 nM, lanes 3 and 8) or polV (UmuC_Y11A) (60 nM, lanes 5 and 10) were performed for 30 sec as described in the Methods section. Part of the template sequence and position of the gel wells and a CPD lesion are indicated to the right of the gel panel. As clearly observed, when present in a 10-fold excess (similar to SOS induced conditions), pol IV inhibits TLS catalyzed by pol V.</p

Effect of deleting <i>rnhA</i>, <i>rnhB</i> and/or <i>uvrA</i> alone, or in various combinations, on the extent of <i>umuC</i>_Y11A-dependent spontaneous mutagenesis in <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains.

<p>We constructed a series of isogenic <i>recA730 lexA</i>(Def) Δ<i>dinB</i> Δ<i>umuDC</i> strains with Δ<i>rnhA</i>, Δ<i>rnhB</i> or Δ<i>uvrA</i> alleles alone, or in various combinations and assayed pol V-dependent spontaneous mutagenesis in strains harboring plasmids expressing wild-type pol V or <i>umuC</i>_Y11A by assaying reversion of the <i>hisG4</i> (ochre) allele. The data plotted on the graph represent the <i>relative</i> extent of spontaneous mutagenesis in cells expressing <i>umuC</i>_Y11A calculated as a percentage of the spontaneous mutagenesis in cells expressing wild-type pol V. All experiments were performed in triplicate. Standard errors were calculated taking into account variability in spontaneous mutagenesis in each strain. Data for the wild-type, Δ<i>rnhA</i>, Δ<i>rnhB and the</i> Δ<i>rnhA</i> Δ<i>rnhB</i> strains were taken from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003878#pgen.1003878-McDonald1" target="_blank">[11]</a> and are shown for comparison. As clearly observed, in contrast to the Δ<i>rnhA or</i> Δ<i>uvrA</i> strains, which exhibit roughly the same extent of <i>umuC</i>_Y11A-dependent mutagenesis as the wild-type strain, the Δ<i>rnhB</i> strain exhibited significantly higher levels of spontaneous mutagenesis, suggesting that the <i>rnhB</i>-encoded RNase HII repair pathway is the primary defense against errant ribonucleotide incorporation in <i>E.coli</i>. However, in contrast to the <i>rnhB</i>⁺ strains, deletion of either <i>rnhA</i> or <i>uvrA</i> in the Δ<i>rnhB</i> strain background leads to a further increase in <i>umuC</i>_Y11A-dependent spontaneous mutagenesis, suggesting that both enzymes participate in back-up pathways of ribonucleotide repair. Furthermore, <i>umuC</i>_Y11A-dependent spontaneous mutagenesis in the Δ<i>rnhA</i> Δ<i>rnhB</i> Δ<i>uvrA</i> triple mutant strain is actually higher than in the isogenic strain expressing wild-type pol V. These data imply that all major pathways specific for ribonucleotide repair are blocked in this strain background.</p

NER cleavage reaction products generated using various DNA-RNA-DNA hybrid substrates.

<p><b>I</b>; Cartoon of the synthetic substrates used in the <i>in vitro</i> assays, with the sites of incision and expected product size indicated, along with the DNA sequence containing rNMP(s) and mismatched nucleotides. <b>II</b>; The 50-mer duplexes (10 nM) in which the modified strand was 5′ end-labeled (indicated by *), were incubated with the NER proteins at concentrations of 40 nM (UvrA), 200 nM (UvrB), and 100 nM (UvrC) for 60 min at 55°C in the presence of 1 mM ATP. UvrABC-dependent incision on the fluorescein adducted 50-bp duplex (fT) was used as a control NER activity of the purified proteins (panel A). The DNA duplexes (panel B) as well as DNA-RNA-DNA hybrids either containing a single rAMP (panels C), two consecutive rAMPs (panel D), or five rNMPs (panels E) were assayed. The reaction products were separated under denaturing conditions by 15% polyacrylamide gel electrophoresis (PAGE). The efficiency of UvrABC-dependent incision was determined as a percentage of the radioactivity in the incised products relative to the total signal of the substrate (% inc.). The data below the gels are mean values calculated from at least two independent experiments. The DNA sequence containing rNMP(s) is shown alongside the gels where DNA and RNA are represented by uppercase and lowercase letters, respectively. Orange arrows indicate the cleavage sites. The red bracket indicates spontaneous ribonucleotide cleavage. UDS, refers to the undamaged strand, and DS, is damage/ribonucleotide-containing strand. The <i>in vitro</i> assays reveal that the NER proteins incise the DNA backbone 8 base pairs 5′ of ribonucleotides and that the reactions are stimulated by base mispairs.</p