GENOMIC INSTABILITY

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Summary of work The protein p21 (Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin dependent kinases (CDKs) and cyclins. P21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA) which is an auxiliary factor for polymerase delta;. In addition to its role on replication, PCNA is clearly implicated in the repair resynthesis step of nucleotide excision repair (NER). Since PCNA particpates in both DNA repair and replication, it has been speculated that p21 might also regulate NER through its interaction with PCNA. Previous studies on the role of p21 on NER have yielded contradictory results that make it difficult to reach a general consensus with regard to the precise role of p21 in NER. We have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain or the PCNA-binding domain of the protein. The results show that the C-terminal domain of the p21 protein, which binds to PCNA, inhibits NER both in vitro and in vivo. A 50% percent inhibition of in vitro NER occurred at a ratio of 50:1 p21 C-terminus to PCNA monomer. This is a specific function of the C-terminal end of p21 since the N-terminal of the protein had no effect on nucleotide excision repair. Our results suggest that the inhibition occurs at the resynthesis step of the repair process. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA since it is relieved by addition of purified PCNA protein. We conclude that the effect of p21 on DNA repair occurs at much higher concentrations than its effect on DNA replication. P21 might serve as an intracellular regulator of these two processesWe are currently exploring if P21 also participates in another major DNA repair pathway, that of Base excision repair (BER). PCNA is involved in the long patch BER repair and we expect that p21 will influence that pathway as well. Further, it is being examined whether p21 also affects the DNA repair in mitochondria, where base excision also operates.