Rev1 forms a complex with Pol31 and Pol32.

<p>(A) Purity of the proteins. We analyzed 0.5 µg of each protein on a 10% denaturing SDS-polyacrylamide gel. Molecular mass standards are shown on the right. (B) GST pull-down of the purified proteins. GST–Pol32 immobilized on glutathione–Sepharose beads was incubated with purified Pol31 and Rev1. After washing, bound proteins were eluted with glutathione. Aliquots of each sample, taken before addition to the beads (L), from the flow-through fraction (F), from the last wash (W), and from the glutathione-eluted proteins (E), were analyzed on 10% SDS-polyacrylamide gel (lanes 1–4). The results for the control experiment using GST instead of GST–Pol32 are shown in lanes 5–8. Molecular mass standards are shown on the right.</p

Model for polymerase exchange at a DNA damage site.

<p>DNA damage stalls the replication complex and triggers the ubiquitylation of PCNA by Rad6–Rad18 at the stalled fork. Monoubiquitylated PCNA promotes TLS, for which to occur first Pol3 is removed from the stalled complex through ubiquitylation-mediated proteasomal degradation, assisted by Def1. A TLS polymerase takes over the place of Pol3, and together with Pol31 and Pol32 carries out lesion bypass. After the deubiquitylation of PCNA, Pol3 regains its place at the replication complex, and normal replication resumes. For simplicity, only half of the replication fork is shown. The DNA damage site on the template strand is marked by a black diamond symbol.</p

UV-dose–dependent degradation of Pol3.

<p>Cultures of <i>mms2</i> cells were synchronized by α-factor and irradiated with increasing doses of UV, as indicated. After released back to growth, 1 ml of cells was collected at the indicated time points, and cell extracts were analyzed by Western blotting. Anti-HA detected HA-tagged Pol3, and PGK served as a loading control. The level of Pol3 relative to PGK is shown at the bottom of each panel.</p

Pol3 degradation depends on <i>RAD6</i> and <i>DEF1</i>.

<p>Cultures were synchronized by α-factor, UV-irradiated with 150 J/m², and released back to growth media. Proteins from whole cell extracts, prepared from 1 ml of cell culture collected at the indicated time points after UV treatment, were analyzed by Western blotting. Anti-HA antibody was used to detect HA-tagged Pol3 (A to E), Pol31 (F), or Pol32 (G). Cell cycle progression was monitored by Clb2 cyclin levels, and PGK served as a loading control. The level of Pol3 relative to PGK is shown at the bottom of each panel.</p

Alkaline BrdU comet PRR images of HCT116(RAD18^−/−) cells after UV irradiation.

<p>(<b>A</b>) UV irradiated (20 J/m²) cells were allowed to recover for 6 hour. Representative images of UV treated cells display highly discontinuous, fragmented tails. The modified BrdU comet assay allowed proper differentiation between comet head and tail, which is essential for precise quantitation. Following the BrdU comet assay, the incorporated BrdU was identified by anti-BrdU primary antibody and cells were detected using Alexa Fluor 488 conjugated secondary antibody (green, panel I). Ethidium bromide counterstaining of the same cells (red, panel II) and merged images (yellow, panel III) are shown. (<b>B</b>) BrdU immunostaining detects S-phase cells without synchronisation. HCT116(RAD18^−/−) cell were UV irradiated (40 J/m²) and allowed to recover for 6 hour. Staining was carried out as described in (A). Arrows indicate the non-S-phase cells which has not been stained with anti-BrdU antibody but only with ethidium bromide.</p

<i>DEF1</i> participates in the <i>REV3</i> branch of the <i>RAD6</i>-governed DNA damage tolerance.

<p>(A–E) Epistatic analysis of <i>DEF1</i> with mutants of the different branches of the <i>RAD6</i> pathway upon UV irradiation. Standard deviations are indicated. (F–J) Epistatic analysis of the same mutants upon MMS treatment. (K, L) Genetic interactions of <i>RAD30</i> with <i>MMS2</i> and <i>REV3</i> upon MMS treatment. All experiments were repeated at least three times.</p

Alkaline BrdU comet PRR assay images of untreated and UV-irradiated HeLa cells representing the progression of replication as detailed in Figure 1.

<p>The cells were pulse labelled with BrdU before irradiation with 20 J/m² UV-C or mock treatment. After the indicated time (0–6 h) single-stranded DNA fragments were separated from matured DNA by alkaline single cell electrophoresis followed by immunostaining using fluorescent anti-BrdU antibody (green) or staining with ethidium-bromide (red). The much higher sensitivity of the BrdU comet assay as compared to the basic comet assay was demonstrated by showing the anti-BrdU stained and ethidium bromide counterstained images of the same cells.</p

Dose dependent inhibition of replication progression caused by UV as detected by BrdU comet PRR assay.

<p>Cells were BrdU pulse labelled and mock treated or irradiated with UV and replication fork progression was followed by alkaline BrdU comet PRR assay. BrdU was detected by immunostaining, and quantitative measurement of comet tail DNA was done by Komet5 software. The % comet tail DNA, as a measure of discontinuity was illustrated. (<b>A</b>) Dose-effect curve of HeLa cells after 4 h recovery time. (<b>B</b>) Dose-effect curve of HCT116(wt), HCT116(Rad18^−/−)cell lines after 6 hour recovery. Each data point represents the mean of three independent experiments. Error bars indicate standard deviations. (<b>C</b>) Representative alkaline BrdU comet PRR images as revealed by anti-BrdU immunostaining of HCT116(wt) cells irradiated with increased UV dose as indicated followed by 6 hour recovery. (<b>D</b>) As (C) but HCT116(Rad18^−/−) cells were used instead of wild type cells.</p