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

<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

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

Two Scaffolds from Two Flips: (α,β)/(β,γ) CH₂/NH “Met-Im” Analogues of dTTP

Novel α,β-CH₂ and β,γ-NH (<b>1a</b>) or α,β-NH and β,γ-CH₂ (<b>1b</b>) “Met-Im” dTTPs were synthesized via monodemethylation of triethyl-dimethyl phosphorimido-bisphosphonate synthons (<b>4a</b>, <b>4b</b>), formed via a base-induced [1,3]-rearrangement of precursors (<b>3a</b>, <b>3b</b>) in a reaction with dimethyl or diethyl phosphochloridate. Anomerization during final bromotrimethylsilane (BTMS) deprotection after Mitsunobu conjugation with dT was avoided by microwave conditions. <b>1a</b> was 9-fold more potent in inhibiting DNA polymerase β, attributed to an NH-group interaction with R183 in the active site

β,γ-CHF- and β,γ-CHCl-dGTP Diastereomers: Synthesis, Discrete ³¹P NMR Signatures, and Absolute Configurations of New Stereochemical Probes for DNA Polymerases

Deoxynucleoside 5′-triphosphate analogues in which the β,γ-bridging oxygen has been replaced with a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base-selection mechanisms. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a general solution to this long-standing problem with four examples of β,γ-CXY dNTP diastereomers: (<i>S</i>)- and (<i>R</i>)-β,γ-CHCl-dGTP (<b>12a-1</b>/<b>12a-2</b>) and (<i>S</i>)- and (<i>R</i>)-β,γ-CHF-dGTP (<b>12b-1</b>/<b>12b-2</b>). Central to their preparation was conversion of the prochiral parent bisphosphonic acids to the P,C-dimorpholinamide derivatives <b>7</b> of their (<i>R</i>)-mandelic acid monoesters, which provided access to the individual diastereomers <b>7a-1</b>, <b>7a-2</b>, <b>7b-1</b>, and <b>7b-2</b> by preparative HPLC. Selective acidic hydrolysis of the P–N bond then afforded “portal” diastereomers, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H₂ (Pd/C) gave the four individual diastereomeric nucleotides <b>12</b>, which were characterized by ³¹P, ¹H, and ¹⁹F NMR spectroscopy and by mass spectrometry. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH ∼10 the diastereomer pairs <b>12a</b>,<b>b</b> exhibit discrete P_α and P_β ³¹P resonances. The more upfield P_α and more downfield P_β resonances (and also the more upfield ¹⁹F NMR resonance in <b>12b</b>) are assigned to the <i>R</i> configuration at the P_β-CHX-P_γ carbons on the basis of the absolute configurations of the individual diastereomers as determined from the X-ray crystallographic structures of their ternary complexes with DNA and polymerase β

Mapping Functional Substrate–Enzyme Interactions in the pol β Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs

We report high-resolution crystal structures of DNA polymerase (pol) β in ternary complex with a panel of incoming dNTPs carrying acidity-modified 5′-triphosphate groups. These novel dNTP analogues have a variety of halomethylene substitutions replacing the bridging oxygen between Pβ and Pγ of the incoming dNTP, whereas other analogues have alkaline substitutions at the bridging oxygen. Use of these analogues allows the first systematic comparison of effects of 5′-triphosphate acidity modification on active site structures and the rate constant of DNA synthesis. These ternary complex structures with incoming dATP, dTTP, and dCTP analogues reveal the enzyme’s active site is not grossly altered by the acidity modifications of the triphosphate group, yet with analogues of all three incoming dNTP bases, subtle structural differences are apparent in interactions around the nascent base pair and at the guanidinium groups of active site arginine residues. These results are important for understanding how acidity modification of the incoming dNTP’s 5′-triphosphate can influence DNA polymerase activity and the significance of interactions at arginines 183 and 149 in the active site