Detection of Chromatin-Bound PCNA in Cultured Cells Following Exposure to DNA-Damaging Agents

It has been a decade since proliferating cell nuclear antigen (PCNA) was found to be essential for DNA replication as an auxiliary protein of DNA polymerase δ (1 ,2 ; reviewed in 3 ,4 ). There is compelling evidence that a homotrimer of PCNA forms a toroidal clamp around duplex DNA (5 –7 ), an association catalyzed by replication factor C (RF-C) in an ATP-dependent manner (8 ). The PCNA clamp binds to polymerase δ and stimulates its processivity by sliding along the DNA template. PCNA similarly enhances the activity of another DNA polymerase, ε, under certain conditions in vitro (9 ). PCNA is now also known to be an indispensable factor in nucleotide excision repair (NER) (10 ) and in an alternative pathway of base excision repair (11 ,12 ). Thus, detection of chromatin-associated PCNA in cells exposed to DNA-damaging agents is indicative of DNA repair activity, provided the cells are quiescent or at least in G1 or G2 phase. One may, however, encounter a problem in detecting such a PCNA clamp, because chromatin-unbound PCNA is also present within nuclei.