Figure 5. Rfc3 is required for the unloading of PCNA from closed-circular DNA.
(A) Depletion efficiency of xRfc3 from NPE. The depletion efficiency was estimated to be >98%. (B) hPCNA loaded onto immobilized pMM2AT (carrying no mismatch) was incubated in NPE described in (A). The efficiency of nick ligation in the PCNA loading reaction was estimated to be ~89%.
Figure 5—figure supplement 1. Specific detection of hPCNA by a monoclonal antibody.
The hPCNA monoclonal antibody used in this study does not detect xPCNA. 10 ng each of recombinant xPCNA and hPCNA were separated using 4–15% SDS-PAGE. Recombinant proteins were visualized using silver nitrate or transferred onto PVDF membranes. The membrane strip was probed with hPCNA antibody. hPCNA monoclonal antibodies do not detect recombinant xPCNA even when the membrane was exposed for longer periods (lanes 3–6).