Analysis of Rev3-PCNA and Rev7-PCNA interactions in cells deleted for <i>UBP10</i>.

<p>(A) Rev3 (DNA polymerase ζ catalytic subunit) interacts with PCNA similarly in wild-type and <i>ubp10Δ</i> cells. Co-immunoprecipitation assay showing physical interaction between Rev3-myc and PCNA-FLAG. PCNA-FLAG was immunoprecipitated from asynchronously growing or 0.02% MMS-treated cells (as indicated), blots were incubated with α-myc (to detect Rev3) or α-FLAG (to detect PCNA). As indicated the strains used in this assays were <i>REV3-myc POL30-FLAG</i>, <i>REV3-myc POL30-FLAG ubp10Δ</i> and single tagged <i>POL30-FLAG</i> or <i>REV3-myc</i> controls. Whole cell extracts (WCE) and mock Ip controls are also shown as indicated. (B) The interaction of PCNA with Rev7 (an accessory subunit of DNA polymerase ζ) is reduced in cells deleted for <i>UBP10</i>. Co-immunoprecipitation assay of Rev7-myc and PCNA-FLAG. PCNA-FLAG was immunoprecipitated from asynchronously growing or 0.02% MMS-treated cells (as indicated), blots were incubated with α-myc (to detect Rev7) or α-FLAG (to detect PCNA). As indicated, the key strains used in this assays were <i>REV7-myc POL30-FLAG</i> and <i>REV7-myc POL30-FLAG ubp10Δ.</i> Appropriate single tagged, input (WCE) and mock Ip controls are shown. (C) Deletion of <i>UBP10</i> alters the interaction of PCNA with Rev7. To assure that deletion of <i>UBP10</i> reduced significantly Rev7-PCNA interaction, <i>UBP10</i> was deleted in the <i>REV7-myc POL30-FLAG</i> strain used in B. Five different <i>REV7-myc POL30-FLAG ubp10Δ</i> deletion strains and a <i>REV7-myc POL30-FLAG</i> control were used in the co-immunoprecipitation analysis. PCNA-FLAG was immunoprecipitated from asynchronously growing cells, blots were incubated with α-myc (to detect Rev7) or α-FLAG (to detect PCNA). The strains used in this assays were either <i>REV7-myc POL30-FLAG (1)</i> or <i>REV7-myc POL30-FLAG ubp10Δ</i> (2a, 2b, 2c, 2d and 2e). Input whole cell extracts (WCE) and mock Ip controls are shown. Note that similar amounts of Rev7 are present in whole cell extracts of <i>REV7-myc POL30-FLAG</i> (1) and <i>REV7-myc POL30-FLAG ubp10Δ</i> (2c) cells.