(A) Mapping of binding domain of SENP6 with RPA70. HEK-293 cells were co-transfected with 2 µg of c-Myc-tagged RPA70 and 2 µg of FLAG-tagged full-length or truncated SENP6, as indicated. Tagged RPA70 was precipitated by anti-c-Myc affinity matrix and immunoblotted with anti-FLAG antibodies (upper panel). Immunoblotting with anti-FLAG (middle panel) and anti-c-Myc (lower panel) antibodies were performed to ensure that equivalent amounts of truncated SENP6 were present in the lysates before immunoprecipitation or to use as the loading control for IP Western. The samples of anti-c-Myc (lower panel) came from IP. FL: full length, T1: SENP6(1–974); T2: SENP6(1–777); T3: SENP6(1–629); T4: SENP6(1–379).
(B) Co-localization of RPA70 and SENP6 in S-phase cells. HeLa cells were synchronized in mitosis following a previously described procedure (Dimitrova et al., 1999). Metaphase cells were released in the next cell cycle and aliquots of the cells were either collected 4 hr later (G1 phase) or synchronized at the G1/S-phase border through a double-thymidine block as described in Methods and subsequently released in free medium for 3 h (S-phase) and 9 hr (G₂/M phase). The cells in G1 and S phases were Triton-extracted, fixed, and stained for indicated antibodies. For cells in G₂/M, cells released for 9 hr from second thymidine block were trypsinized, CSK-extracted, fixed, and cytospinned onto cover slide to perform immunostaining. Representative images from different stages of cell cycle were shown. SENP6 (green), RPA70 (red), or PCNA (red). Co-localization of SENP6 with RPA70 or PCNA is visible in yellow.
(C) SENP6 associates with RPA70 during S phase.
HeLa cells were synchronized by double-thymidine block and harvested at indicated time. Endogenous SENP6 was co-precipitated with anti-RPA70 polyclonal antibodies and immunoblotted with anti-SENP6 antibodies (upper panel). Input of precipitated RPA70 was standardized with anti-RPA70 monoclonal antibodies (middle panel). Endogenous SENP6 in the WCL was confirmed by immunoblotting with anti-SENP6 monoclonal antibodies (lower panel). Inserted box shows the cell cycle profile at different time after release from double-thymidine block.

(A, B) RPA70 SUMOylation in response to CPT. Asynchronous HeLa cells were treated with different concentration of CPT (A) or collected at indicated time points after CPT treatment (B). The whole cell lysates were analyzed by immunoblotting with anti-RPA70, anti-γ-H2AX or anti-actin antibodies.
(C) CPT induces dissociation of RPA70 from SENP6. Endogenous RPA70 was immunoprecipitated by rabbit anti-RPA70 polyclonal antibodies or control IgG and immunoblotted with anti-SENP6 (upper panel) or anti-RPA70 (middle panel) monoclonal antibodies. WCL was blotted with anti-SENP6 antibodies (lower panel).
(D) CPT regulates SENP6/RPA70 association and RPA70 SUMOylation on the chromatin. Chromatin fractions of HeLa cells treated with CPT for different times were prepared as described in Methods. Chromatin-associated SENP6 and RPA70 were released by micrococal nuclease and were immunoprecipitated with anti-SENP6 antibody or control IgG and immunoblotted with anti-RPA70 (upper panel) or anti-SENP6 antibody (second panel). Chromatin fraction was also immunoblotted with anti-SENP6 (third panel), anti-RPA70 (fourth panel), or anti-Histone H3 (lowest panel).

(A) SUMOylation of RPA70 enhances its association with Rad51. COS-1 cells were co-transfected with FLAG-tagged RPA70-UBC9 or FLAG-tagged RPA70(ΔSUMO)-UBC9 and HA-tagged SUMO-2 plasmids. FLAG-tagged protein was purified by FLAG-EZview™ Red Affinity Gel. C-terminal fused UBC9 was removed by thrombin (see Methods). The precipitates were separated by SDS-PAGE stained with coomassie blue (left lane). The same samples were serially diluted ((1/4(lane 1):1/2(lane 2):1(lane3)) and applied for western blot by probing with HRP-conjugated anti-FLAG antibody (middle lane) or for far-western blot (right panel) that FLAG-immunoprecipitation was first subjected to SDS/PAGE, and then transferred to a membrane. The membrane-bound proteins were re-natured, incubated with recombinant Rad51, and then probed with anti-Rad51 antibodies. Two lower bands are RPA32 and RPA14. *, indicates non-specific bands. Relative intensity of the FLAG-RPA70 or FLAG-RPA70-SUMO2 regions of the coomassie blue gel (left panel) was determined by densitometry. FLAG-RPA70 was set as 100%. Error bar indicates s.d. for the results of three densitometry measurements. For the middle and right panels, the average relative intensity was determined by three measurements of the different serial dilutions. Error bars indicate s.d. for three relative intensity measurements.
(B) SUMO-2 interacts with Rad51. Recombinant Rad51 (500ng) was incubated with GST (500ng) and GST-SUMO-2 (500ng) in 0.5ml binding buffer for 2 hours at 4°C. 10 µl Glutathione-Sepharose™-4B beads were used for pull-down experiments. The precipitates were immunoblotted with anti-Rad51 or anti-GST antibodies. Ubc9 has previously been shown to be involved in binding of SUMO-2 to Rad51. However, we and others showed that SUMO-2 can directly bind to Rad51 in the absence of UBC9 (Ouyang et al., 2009).

Association of SENP6 with RPA70 during normal S phase maintains RPA70 in a hypo-SUMOylated state. This is shown by a dashed line to indicate that SENP6 and RPA70 association is not stoichiometric. With CPT treatment or exposure to IR, SENP6 becomes dissociated from RPA70. SUMOylation of RPA70 then leads to enhanced recruitment of Rad51 to initiate HR.

(A) SENP6-knockdown induces RPA70 SUMOylation. Cell lysates were prepared at 72hr after transfection with indicated siRNAs, separated by SDS-PAGE, and immunoblotted with anti-RPA70 monoclonal antibodies. Two different exposures were shown.
(B) Endogenous RPA70 is modified by endogenous SUMO-2/3 in vivo. SENP6-knockdown was described in Methods. Cell lysate was aliquoted equally for performing immunoprecipitation by control IgG or RPA70 monoclonal antibody. Endogenous SUMOylated RPA70 was immunoblotted with anti-SUMO-1 or anti-SUMO-2/3 antibodies. Loading samples were standardized by blotting with anti-RPA70 monoclonal antibodies.
(C) RPA70 is modified by SUMO-2 in an over-expression system. COS-1 cells were transfected with indicated c-Myc-tagged RPA70 (2.5 ug) and HA-tagged SUMO-2 (0.5 ug) plasmids. Immunoprecipitation was performed with Myc-EZview™ Red Affinity Gel matrix 24hr after transfection and analyzed with c-Myc (upper panel) or HA antibodies (lower panel).
(D) Over-expression of SENP6 de-conjugates SUMOylated RPA70. COS-1 cells were transfected with c-Myc-tagged RPA70 (2.0 µg), HA-tagged SUMO-2 (0.5 µg), and SENP6 (0.5 µg) or SENP6 catalytic site mutant (0.5 µg) plasmids. Tagged RPA70 was precipitated and immunoblotted with anti-HA (upper panel) or anti-c-Myc antibodies (middle panel). SENP6 expression was confirmed by immunoblotting with anti-FLAG antibodies (lower panel). Over-expression of SENP6 may result in non-specific cleavage. However, the true specificity of SENP6 was shown by the siRNA knock down studies shown in Figure 2A and B.
(E) Determination of SUMOylation sites in RPA70. COS-1 cells were transfected with 2.0 µg of c-Myc-tagged RPA70 or its mutants along with HA-SUMO-2 plasmids. Immunoprecipitates of c-Myc-tagged RPA70 were immunoblotted with anti-HA (upper panel) or anti-c-Myc antibodies (lower panel).

(A) Establishment of stable siRNA-resistant FLAG-tagged RPA70 cell cells. This was accomplished by stable expression of FLAG-tagged, siRNA-resistant FLAG-tagged RPA70(WT) or RPA70(ΔSUMO) by infecting HeLa cells with PQCXIP vectors. The expression levels of FLAG-tagged RPA70(WT) or RPA70(ΔSUMO) was similar to endogenous RPA70.
(B) Endogenous RPA70 was knocked down with transfection of 50nM siRNAs and cell lysates were analyzed by RPA70 monoclonal antibodies. Loading samples were standardized by immunoblotting of actin.
(C) Endogenous RPA70 and SENP6 was knocked down in RPA70(WT) or RPA70(ΔSUMO) cell lines. Cells were harvested by trypsinization 72 hr after transfection and analyzed by RPA70 monoclonal antibodies. Loading samples were standardized by immunoblotting of actin. As shown, SENP6 knockdown induces SUMOylation of FLAG-tagged RPA70 only in the RPA70(WT), but not in the RPA70(ΔSUMO) cell lines. There is a slight increase in the upper band in the RPA70(ΔSUMO) in the SENP6 knockdown cells as compared to control. This could be due to the presence of minor SUMOylation site or experimental variations. (D) RPA70(ΔSUMO) was more sensitive to CPT than wild type RPA70(WT). In left panel, endogenous RPA70 was knockdown as described in (B) in HeLa cells that carried siRNA-resistant RPA70 (WT) or RPA70 (ΔSUMO). Knockdown of Rad51AP1 was used as a positive control (right panel). HeLa cells that carried siRNA-resistant RPA70 (WT) or RPA70 (ΔSUMO) were simultaneously transfected with 50nM siRNAs against RPA70 and Rad51AP1 to knock down endogenous RPA70 and Rad51AP1. These cells were trypsinized and 1000 cells were seeded onto 60 mm diameter dishes. After a 12hr attachment period, cells were treated with increasing doses of CPT for 12hr. Cells were subsequently washed twice with PBS and incubated in fresh media for 7–10 days, after which the colonies were fixed, stained and counted. The efficiency of Rad51AP1 knockdown was shown in Figure S4C. Error bars indicate s.d. for the results of triplicate assays.

(A) SUMOylation facilitates Rad51 to displace RPA from ssDNA as measured by a time courses of ATPase activity. Input of FLAG-RPA70 (ΔSUMO) (circle) or a mixture of FLAG-RPA70 and FLAG-RPA70-SUMO-2 (triangle) is shown in right panel. FLAG-tagged recombinant proteins were prepared by immunoprecipitation with FLAG-EZview™ Red Affinity Gel matrix from the lysate of COS-1 cells that were co-transfected with FLAG-RPA70-UBC9 or FLAG-RPA70(ΔSUMO)-UBC9 and HA-SUMO-2 cDNAs, eluted with 3XFLAG peptide, and C-terminal fused-UBC9 was removed by thrombin. The reactions were started by addition of Rad51 protein (2.5 µM) to a preformed complex of 1 µM FLAG-RPA70 (ΔSUMO) or a mixture of FLAG-RPA70 and FLAG-RPA70-SUMO-2 with poly (dT) (1.5 µM). The rate of ATP hydrolysis was calculated from the rate of change in absorbance at 340 nm (see Methods). Error bars indicate s.d. for the results of triplicate assays.
(B) SUMOylation mutant is defective in the recruitment of Rad51 to RPA70 foci following CPT treatment. HeLa cells that carried siRNA-resistant RPA70 (WT) or RPA70(∆SUMO) were transfected with siRNAs against RPA70 to knock down endogenous RPA70. Cells treated by 4 µM CTP for 30min were extracted with CSK buffer at indicated time and stained with rabbit anti-Rad51 and mouse anti-RAP70 antibodies. Only nuclei showing more than 5 clearly co-localized-foci were considered as positive. More than 300 cells per sample were analyzed at each independent experiment. Error bars indicate s.d. for the results of triplicate assays.
(C) SUMOylation mutant causes decrease of SCE events in response to CPT. Cells were treated as described in Methods. 30 metaphases were analyzed. Error bars indicate s.d. for the results of triplicate assays.