Volume 23, Issue 2, Pages 265-271 (July 2006) REV1 Protein Interacts with PCNA: Significance of the REV1 BRCT Domain In Vitro and In Vivo  Caixia Guo, Eiichiro Sonoda, Tie-Shan Tang, Joanne L. Parker, Aleksandra B. Bielen, Shunichi Takeda, Helle D. Ulrich, Errol C. Friedberg  Molecular Cell  Volume 23, Issue 2, Pages 265-271 (July 2006) DOI: 10.1016/j.molcel.2006.05.038 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 The BRCT Domain in REV1 Mediates Its Association with PCNA (A) Anti-Flag M2 agarose affinity gel was incubated with cos7 cell lysates expressing Myc-REV1 and Flag-PCNA fusion proteins. Immunoprecipitates were analyzed by immunoblotting with α-Myc (top) or α-Flag (bottom). Lane 1, input containing 1/75 of the lysates used for immunoprecipitation; lanes 2 and 3, immunoprecipitation with mock antibody (lane 2) and α-Flag antibody (lane 3). (B) MRC5 cells expressing HA-tagged REV1 were UV irradiated and incubated for 7 hr prior to triton extraction and crosslinking. Triton-insoluble proteins were solubilized and immunoprecipitated with α-PCNA or mock (α-Myc) antibody and the retained proteins analyzed by Western blotting with α-HA (top) or α-PCNA (bottom). (C) GST-PCNA pull-down of purified REV1 protein. Recombinant mouse REV1 was incubated with GST or GST-PCNA as indicated. Bound proteins were detected by immunoblotting with α-REV1. Lane 1 contains 1/10 of the REV1 used in the experiments. (D) Cos7 cell lysates expressing HA-tagged full-length and truncated REV1 proteins were pulled down with GST-PCNA fusion proteins as indicated. Bound proteins were detected by immunoblot analysis with α-HA. Abbreviation: TCL, total cell lysates. Lanes 1–4 contain 1/75 of the lysates used in the experiment. (E) Cos7 cell lysates expressing HA-tagged wild-type and BRCT∗ REV1 proteins were incubated with GST-PCNA fusion proteins as indicated. (F) Cos7 cell lysates expressing GFP-tagged wild-type or BRCT∗ REV1 proteins were incubated with GST-PCNA or GST-PCNA-Ub fusion proteins as indicated. Bound proteins were detected by immunoblotting with α-GFP. Molecular Cell 2006 23, 265-271DOI: (10.1016/j.molcel.2006.05.038) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 The BRCT Domain Is Required for the Constitutive Localization of REV1 in Nuclei Foci (A) MRC5 cells were transfected with plasmids encoding full-length or mutants of eGFP-REV1 as indicated. Twenty hours after transfection, cells were UV irradiated (10 J/m2) (bottom) and incubated for 8–16 hr before fixation with paraformaldehyde. The distribution of REV1 or mutants (as indicated) was observed directly by autofluorescence of eGFP. The images show unirradiated (top) or UV-irradiated (bottom) transfected cells. (B) REV1 focus formation after UV radiation. MRC5 cells were transfected and UV irradiated as above and further incubated for 8 hr. The proportion of eGFP-REV1 expressing cells in which the protein was localized in nuclear foci was determined. All experiments were carried out in triplicate. Error bars indicate the standard error of the mean. Molecular Cell 2006 23, 265-271DOI: (10.1016/j.molcel.2006.05.038) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 The BRCT Domain of REV1 Contributes to DNA Damage Tolerance in DT40 and Yeast Cells (A and B) Δrev1 DT40 clones containing wild-type or BRCT∗ mouse REV1 cDNA were established, and their UV and CDDP sensitivities were measured by clonogenic colony survival assay. The data shown are the representative results from three independent transformants, which exhibited almost the same sensitivity. Data points show the average and standard deviation of triplicate experiments for each clone. (C) Quantification of the MMS sensitivities in S. cerevisiae by determination of survival after treatment with 0.1% MMS for varying times. rev1, empty expression vector; REV1, wild-type REV1 allele; and BRCT∗, G193R. Standard deviations were calculated from triplicate measurements. (D) Effect of the BRCT mutation on MMS-induced mutagenesis. The number of canavanine-resistant (can1R) mutants per 106 surviving cells in response to varying doses of MMS was determined by plating aliquots of treated cultures on YPD to determine viable cell numbers and on canavanine-containing plates for measuring the number of mutants. Symbols are used as in (C). Molecular Cell 2006 23, 265-271DOI: (10.1016/j.molcel.2006.05.038) Copyright © 2006 Elsevier Inc. Terms and Conditions