Detection of Chromatin-bound PCNA in Mammalian Cells and Its Use to Study DNA Excision Repair

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Abstract
Compelling evidence indicates that proliferating cell nuclear antigen (PCNA) is an indispensable factor not only in DNA replication but in nucleotide excision repair (NER), alternative pathway of base excision repair (BER), and mismatch repair. The common function of PCNA in each of these is to assist in the initiation of DNA synthesis by providing a scaffolding clamp as a trimer catalyzed by RF-C at the 3′ -OH terminus of a nascent DNA strand, to which DNA polymerase δ or ε can bind. Interestingly, DNA synthesis is reported to be ingeniously inhibited in replication, but not in NER owing to the interaction with CDKN1A (formerly known as p21/WAF1/CIP1). Furthermore, several proteins, XPG, FEN1, and DNA ligase I, recently were shown to competitively bind to the same region of PCNA, the interdomain connector loop, to which DNA polymerase δ or ε also binds. PCNA therefore seems to have a regulatory role in these DNA transactions. The in vitro reconstituted experimental system has been a powerful tool to obtain these lines of evidence, but another approach, immunofluorescence studies, also has been a contributor. In fact, the involvement of PCNA in DNA replication, NER, and BER has for the first time been indicated by a unique method that makes visible only in vivo chromatin-bound PCNA. The usefulness of this method and the importance of cooperative studies done with in vitro and in vivo experimental systems is discussed in terms of DNA excision repair.