Differential kinetics of mutations induced during NER versus replication.

<p><b>2A:</b> Kinetics of rif^R mutation fixation in various strains following a single UV dose. The UV dose was chosen so as to affect survival of the various strains to a similar extent i.e. ≈10%: wt ■(squares): 100 J/m², <i>dinBpolB</i> X (crosses): 35 J/m², <i>uvrA</i> •(dots): 3 J/m². Following irradiation at the indicated UV dose, cells were platted on rif plates at various time points. Rif^R mutations accumulate rapidly in the wild-type strain (t_1/2 ≈ 30 min after irradiation); in contrast the kinetics of appearance of mutants in the <i>uvrA</i> and in the <i>dinBpolB</i> strains are severely delayed (t_1/2 ≈ 80–100 min after irradiation). The plateau reached in the <i>uvrA</i> and <i>dinBpolB</i> strains correspond to ≈ 25–30% and 15–20% of the level reached in the wild-type strain, respectively (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006881#pgen.1006881.g001" target="_blank">Fig 1E</a>). <b>2B</b>: Kinetics of induction of rif^R mutation in a <i>dnaBts</i> thermo-sensitive replication mutant irradiated at 90 J/m² [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006881#pgen.1006881.ref045" target="_blank">45</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006881#pgen.1006881.ref046" target="_blank">46</a>]. At this dose, survival of <i>dnaBts</i> strain is 11.5%. The <i>dnaBts</i> strain quickly stops replication when shifted at 42°C, the non-permissive temperature. Rif^R mutants are induced at similar kinetics at permissive (35°C: Δtriangles) and non-permissive (42°C: ■squares) temperature, implying that most mutations are fixed in a non-replicative manner.</p

Genetic interactions define a mutation pathway that is dependent upon <i>dinBpolB</i> and nucleotide excision repair genes.

<p><b>1A</b>: Rif^R mutation frequencies were determined in various strains in response to UV irradiation. All strains in fig 1A are constructed in the MG1655 background. To account for the intrinsic differences in UV sensitivity among strains, we compared UV doses leading to similar levels of survival: grey bars correspond to UV doses leading to survival levels ranging between 5–15%, for black bars survival levels range between 1–5% survival. It should be stressed that at these UV doses, the SOS response is fully induced in all strains. White bars represent the level of spontaneous mutation frequency, i.e. no UV irradiation. Average values and standard deviations are plotted for three or more independent experiments per strain. <b>1B</b>: The <i>polA1</i> allele data are presented in a separate panel as the background in which this allele resides is w3110. Background w3110 exhibits a ≈2-fold higher UV-induced mutagenic response compared to the MG1655 background at UV irradiation levels leading to similar survival. Grey bars correspond to UV doses leading to survival levels ranging between 5–15%; white bars represent the level of spontaneous mutation frequency, i.e. no UV irradiation. <b>1C</b>: Survival curves of DNA polymerase mutant strains. Wild-type: ■squares; <i>polB</i>: <b>−</b> (horizontal segment); <i>umuDC</i>: Δ triangles; <i>dinB</i>:◆ losanges; <i>dinBpolB</i>: X crosses; <i>dinBpolBumuDC</i>: ★ stars. <b>1D</b>: Survival curves of DNA polymerase mutant strains in an NER-defective background: <i>uvrA</i> •(dots), <i>uvrAdinBpolB</i> ✚ (crosses), <i>uvrAumuDC</i>: <b>−</b> (horizontal segment). <b>1E</b>: UV-induced mutation frequencies plotted as a function of survival in various strains. Wild-type strain ■(squares), <i>uvrA</i> •(dots), <i>dinBpolB</i> X (crosses), <i>uvrAdinBpolB</i> ✚(crosses), <i>umuDC</i> Δ (triangles).</p

Opposing lesion structure model for NER-induced mutagenesis.

<p><b>A</b>: Nucleotide Excision Repair initiates repair at a given lesion via its normal dual incision step creating a 12–13 nt single-stranded gap. At rare occasions a second lesion will be located in the opposite strand within the initial incision gap or in close proximity. In the latter case the second lesion may becomes exposed within the gap following a gap enlargement step possibly triggered by the distortion induced by the second lesion at the double-stranded / single-stranded junction via exonuclease (or helicase) processing. The resulting structure will be referred to as “opposing lesion structure”. Alternatively, gap enlargement may result from the necessity to allow a RecA filament to assemble downstream from the lesion in order to activate Pol V [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006881#pgen.1006881.ref047" target="_blank">47</a>]. The gap-filling process requires the action of Pol IV/Pol II. The mutation “m” opposite the lesion site is fixed by Pol V during the lesion bypass step. <b>B</b>: Mathematical modeling of UV-induced mutagenesis in a wild-type and <i>dinBpolB</i> strains as a function of UV dose (J/m²). The RiM line fits properly the <i>dinBpolB</i> data points •(dots) (regression coefficient R = 0.91). The wild-type data points ■ (squares) fit appropriately the theoretical curve when the region that defines the opposing lesion zone is set to 18 +/-3 nt (i.e. data points within the 15 and 21 nt range). <b>C</b>: Number of lesions “at risk” (LAR) as a function of UV dose (J/m²). The straight line and the quadratic curve represent the number of lesions, within the <i>rpoB</i> target, at risk for inducing mutations during replication (LAR-RiM) or during NER (LAR-NERiM: opposing lesion zone set to 18nt), respectively (see text). At ≈100J/m2, when the two curves cross, an equal number of mutations will result from replication and NER pathways.</p

Plasmid-based assay to show NERiM for oxidative lesions.

<p>How general is NERiM? The involvement of NER in induced mutagenesis was investigated by implementation of a plasmid-borne mutation assay using a different mutagenic treatment, namely oxidative damage induced by treating plasmid DNA with Methylene Blue and visible light (MB+ light). <b>A</b>: Experimental outline: the assay involves a pBR322-derived plasmid that contains a +2 frameshift mutation within its tetracycline resistance gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006881#pgen.1006881.ref058" target="_blank">58</a>]. The assay was shown to monitor true -2 frameshift reversions that restore tetracycline resistacrossnce [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006881#pgen.1006881.ref059" target="_blank">59</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006881#pgen.1006881.ref060" target="_blank">60</a>]. Plasmid DNA is randomly damaged <i>in vitro</i> with MB+ light treatment and introduced into bacteria by transformation. Cells are plated on tetracycline and ampicillin plates to determine the Tet^S-> Tet^R reversion frequency. <b>B</b>: cells are treated by UV irradiation prior to plasmid transformation in order to induce their SOS response. The UV dose is chosen so as to yield a survival of 10–20% (wild-type: 95 J/m²; dinBpolB: 45 J/m²; uvrA: 5 J/m² and uvrAdinBpolB: 4 J/m²). The average and standard deviation of four to six independent determinations are plotted for each strain. Introduction, into wild-type cells, of plasmid DNA treated with MB+light robustly increases the -2 frameshift mutation frequency by two to three orders of magnitude above untreated control plasmid. The mutagenic response is reduced ≈5-fold when the (MB+light) treated plasmid is introduced into either <i>uvrA</i>, <i>dinBpolB</i> or <i>uvrAdinBpolB</i> strains.</p

Occurrence of NERiM in stationary phase <i>E</i>. <i>coli</i> cells.

<p>When irradiated at 200 J/m² in stationary phase the survival of the wt and uvrA strains were equal to 75% and 63%, respectively. <b>A</b>: Induction of rif^R mutations by UV-light at a dose of 200 J/m² (black bar) or in the absence of irradiation (white bar) in stationary phase <i>E</i>. <i>coli</i> cells. <b>B</b>: suppression of UV-induced mutagenesis in stationary <i>E</i>. <i>coli</i> cells that are defective in SOS induction: <i>lexA</i>(Ind) strain.</p

Strains and plasmids.

<p>Strains and plasmids.</p

Mutagenic TLS through AAF within the 3G sequence or the NarI site in COS-7 and XP12RO cells.

<p>Frequencies of frameshift mutations were expressed as the ratio of the targeted mutant blue colonies to total transformants.</p

Analysis of Rad18 dependence of the G-AAF bypass in HCT116 cell-free extracts.

<p>HCT116 cell-free extracts, wild-type (WT) and Rad18−/− (20 mg), were incubated 30 min at 37°C in the presence of 10 fmoles of AAF-modified substrates either at the 3G sequence or at the NarI site in a final volume of 6.25 ml, as indicated. The samples were analysed by electrophoresis through a 8% denaturing polyacrylamide gel. L-1 is a product generated if synthesis is blocked one nucleotide before and opposite the lesion, respectively. TLS0 and TLS-1 or –2 are products from TLS via non-slipped and slipped intermediates, respectively.Quantitative analysis of experiments with two independent extracts are presented.</p

Analysis of Ub-PCNA dependence of G-AAF bypass in MRC5 cell-free extracts.

<p>Mock depleted (M) or PCNA depleted MRC5 cell extracts (20 mg) were incubated 30 min at 37°C in the presence of 10 fmoles of unmodified or modified substrates in a final volume of 6.25 ml, as indicated. Recombinant wild-type (WT) or mutated K164R PCNA (60 ng) was added to the reactions, as indicated. Aliquotes of the samples were analysed either by Western blot with an anti-PCNA antibody (panel A) or by 8% denaturing polyacrylamide gel electrophoresis (panel B). L-1 is a product generated if synthesis is blocked one nucleotide before the lesion. TLS0 and TLS-1 or –2 are products from TLS via non-slipped and slipped intermediates, respectively. FL are Full Length products.</p