Optimal reaction conditions for the 3′–5′ exonuclease activity of the Ape2

<p><b>Copyright information:</b></p><p>Taken from "Human Ape2 protein has a 3′–5′ exonuclease activity that acts preferentially on mismatched base pairs"</p><p>Nucleic Acids Research 2006;34(9):2508-2515.</p><p>Published online 10 May 2006</p><p>PMCID:PMC1459411.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The 3′–5′ exonuclease activity of GST–Ape2 was assayed under various reaction conditions using a partial DNA duplex (S3) (10 nM) in which the 5′-labeled oligonucleotide contained a 3′-recessed terminus. () Metal ion dependence. The first lane contains reaction mixture without any metal ions in the reaction buffer. Reactions were carried in the presence of 8 mM MgCl (lanes 2–4) or 0.5 mM MnCl (lanes 5–7) and increasing concentrations of Ape2 as indicated. () Graphical representation of results in (A); () NaCl concentration dependence. The first lane contains reaction without any Ape2 protein. () Graphical representation of results in (C); () pH dependence. () Graphical representation of results in (E)

TAP of Rad5 and its complexes.

<p>The final elution fractions of TAP purification of whole cell extracts from control and MMS-treated cultures were analyzed by SDS PAGE. Purified proteins are designated on the right, and the molecular mass markers are indicated on the left. The asterisk denotes a nonspecific band.</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

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

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

UV-induced degradation of Pol3 is mediated by the proteasome and is triggered by its Def1-dependent polyubiquitylation.

<p>(A) The <i>rpn7-3</i> mutant is deficient in degrading Pol3. Experiments were done as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001771#pbio-1001771-g005" target="_blank">Figure 5</a>, except that for the inactivation of the proteasome, cells were shifted to 37°C 2 h before α factor treatment, and after 3 h of synchronization, irradiated with 200 J/m². Anti-HA antibody was used to detect HA-tagged Pol3. 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. (B) Def1 assists Pol3 polyubiquitylation. Polyubiquitylated proteins from cell extracts prepared from 100 ml of cell culture before and 20 and 30 min after UV irradiation were bound to NiNTA agarose (Qiagen), and Pol3 was identified in the bound fraction with HA antibody (upper panels). The PGK and Pol3 levels in the extracts before adding to the beads are also shown, and the bound fractions were probed with anti-ubiquitin antibody (lower panels). The applied UV dose was 150 J/m².</p