Fig 2: Vpr depletes Exo1 by a proteasome-dependent mechanism that is independent of CCNF and an ATR controlled checkpoint. (A) Vpr targets Exo1 for proteasome-dependent degradation. U2OS-iH1Vpr (iH1vpr) and control U2OS cells (mock) were treated, or not, with doxycycline (100 ng/ml) to induce Vpr expression in the absence or presence of the MG132 proteasome inhibitor (1 μg/ml) for 24 h, and cell extracts were analyzed by immunoblotting for the indicated proteins. FLAG-tagged Vpr was detected with an α-FLAG antibody. TFIID provided a loading control. (B) Exo1 depletion by Vpr is not mediated by CCNF. U2OS-iH1Vpr and control U2OS cells were subjected to RNAi targeting CCNF (lanes F) or nontargeting RNAi (lanes NT). Two days later, the cells were treated with doxycycline (100 ng/ml) and/or hydroxyurea (HU; 3 mM) for 24 h, and extracts were analyzed by immunoblotting for Exo1, Vpr, CCNF, total CHK1, and S345-phosphorylated CHK1 (CHK1P). Tubulin (Tub) provided a loading control. (C) Vpr-mediated Exo1 depletion is independent of the induction of the ATR-controlled checkpoint. U2OS-iH1Vpr and control U2OS cells were induced with doxycycline (100 ng/ml) in the presence or absence of the VE-821 ATR inhibitor (2 µM) for 24 h. Cell extracts were analyzed by immunoblotting for the indicated proteins. Tubulin provided a loading control. (D) VE-821 inhibits CHK1 phosphorylation. U2OS cells were cultured in the presence of hydroxyurea (3 mM) in the absence or presence of VE-821 (2 µM) for 24 h. Total and S345-phosphorylated CHK1 levels in cell extracts were revealed by immunoblotting. Lamin B1 (Lamin) provided a loading control.

Fig 3: Exo1 depletion alone does not account for the ability of Vpr to arrest cells in the G2 cell cycle phase. U2OS-iH1vpr cells were subjected to RNAi with siRNAs targeting two distinct regions in Exo1 mRNA (siExo1#1 and #2) or a nontargeting RNAi (siNT). Twenty-four hours postinitiation of RNAi, doxycycline (100 ng/ml) was added, or not, to the culture medium, and 24 h later, the cells were harvested for immunoblot analysis of Exo1 levels (A) or cultured in the presence of ethynyl deoxyuridine (EdU) for 30 min to label S-phase cells actively replicating their DNA and then harvested for flow cytometry analysis of cell cycle profiles (B). (B) The percentage of cells in G1, S, and G2/M phase are indicated. The abscissa is DNA content, revealed by 7AAD staining, shown on a linear scale. The ordinate is DNA synthesis, revealed by incorporation of EdU, shown on a logarithmic scale.

Fig 4: BRCA2-deficient cells accumulate single-stranded DNA lesions in G2. a ?H2AX foci analysis in early mitotic cells. Cells were untreated, or treated with RO-3306 (10 µM, 24 h) to delay mitotic entry, and released for 1 h before analysis of ?H2AX and FANCD2 foci pairs in early mitotic cells. Representative images are shown (left). Analysis is by an unpaired two-tailed t-test. n = 3. Scale bars 10 µm. b–g Cells treated with the indicated siRNAs or mirin (50 µM, 5 h) were incubated with EdU for 30 min and then analyzed for ?H2AX and RPA foci in G2 cells (EdU–, 2N DNA content). Representative deconvolved images are shown (b). Quantification of ?H2AX foci (top, c–g) and RPA+ ?H2AX foci (bottom, c–g) for BRCA2-deficient cells (c), cells transfected with siRNAs (d, RAD51; e SMARCAL1; f EXO1 and DNA2), and cells treated with mirin (g) are shown. n = 3. Scale bars 10 µm. h, i Cells were incubated with EdU as in b before analysis of ?H2AX and pCHK2-T68 (h) or pATM-S1981 (i) foci in G2 cells. Representative deconvolved images with magnified inset highlighting foci co-localization are shown (left in each panel). n = 3. Scale bars 10 µm. Error bars s.d. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (unpaired two-tailed t-test)

Fig 5: Vpr interacts with the Exo1 C-proximal region that binds MSH2. (A) Schematic representation of the Exo1 protein, the tested Exo1 mutants, and their effects on Vpr-mediated depletion of Exo1 levels. Functional organization of the two Exo1 isoforms (Exo1-L and -S) and Exo1 deletion mutants, including location of the nuclease domain, native nuclear localization signal (NLS), and regions mediating binding with the MSH2, MSH3, and MLH1 mismatch repair proteins are shown. A PIP box, located within the MSH2 binding domain, is represented by a red rectangle. The positions of the first and last amino acid residues are indicated for each deletion mutant. The residue 1 to 846 (PIP) variant is a full-length Exo1L with 788QIKLNELW→788AIKANEAA PIP box mutations. The region of Exo1 that contributes to Vpr sensitivity is boxed. Exo1 deletion variants lacking the native NLS were tagged with the SV40 NLS (NLS) at their N terminus, as indicated. (B to D) Vpr interacts with an Exo1 region that comprises the PIP box and mediates binding to MSH2. FLAG-tagged Exo1 and its variants were transiently expressed alone (–) or coexpressed with FLAG-tagged HIV-1 NL4-3 Vpr (+) in HEK293T cells. Cell lysates were prepared 24 h posttransfection, and Exo1 and Vpr levels were revealed by immunoblotting with α-FLAG antibody. An asterisk indicates a nonspecific background band. (E) Vpr targets the PIP box of Exo1 to directly load it onto the CRL4DCAF1 E3. A recombinant MBP-Exo1 fusion protein containing Exo1 residues 749 to 805 [MBP-Exo1(749–805)] or residues 749 to 784 [MBP-Exo1(749–784)] were subjected to in vitro ubiquitination assays with the CRL4DCAF1 or Vpr-CRL4DCAF1 E3 ligase. The reaction mixtures were separated by SDS-PAGE and analyzed by immunoblotting with anti-MBP antibody.