Determination of the Epitope of an Inhibitory Antibody to Proliferating Cell Nuclear Antigen

Proliferating cell nuclear antigen (PCNA) is an essential component for the normal processive DNA synthesis by DNA polymerase δ and is also required for DNA excision repair. Human PCNA autoantisera has been shown to inhibit the function of PCNAin vitro,in contrast to induced antibodies. A monoclonal IgG2PCNA antibody, 74B1, that effectively inhibited DNA replicationin vitrowas identified. The inhibitory effect was dose dependent and using synthetic overlapping peptides of PCNA the 74B1 epitope was mapped to aa 121–135, a region of the PCNA protein containing the interdomain connector implicated in intermolecular interactions. Interestingly, a neighboring and partly overlapping peptide, aa 111–125, contains an immunodominant region recognized by a number of monoclonal PCNA antibodies with no inhibitory effect on DNA synthesis. The 74B1 antibody is a potentially useful antibody in future studies of PCNA and its interaction in complexes associated with cell cycle progress, DNA replication, and DNA repair.