The C-terminal domain of p21 inhibits nucleotide excision repair in vitro and in vivo

The protein p21(Cip1,) (Waf1,) (Sdi1) is a potent inhibitor of cyclin-depen dent kinases (CDKs). p21 can also block DNA replication through its interac tion with the proliferating cell nuclear antigen (PCNA), which is an auxili ary factor for polymerase delta. PCNA is also implicated in the repair resy nthesis step of nucleotide excision repair (NER). Previous studies have yie lded contradictory results on whether p21 regulates NER through its interac tion with PCNA. Resolution of this controversy is of interest because it wo uld help understand how DNA repair and replication are regulated. Hence, 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 (N term inus) or the PCNA binding domain (C terminus) of the protein. In the in vit ro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studi es, we used intact and permeabilized cells. The results show that the C ter minus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vi tro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates D NA repair and replication, with repair being much less sensitive to inhibit ion than replication. Our in vivo results suggest that the inhibition occur s at the resynthesis step of the repair process. It also appears that preas sembly of PCNA at repair sites mitigates the inhibitory effect of p21. We f urther demonstrate that the inhibition of DNA repair is mediated via bindin g of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and th e inhibition of DNA repair by the C terminus of p21 was relieved by the add ition of purified PCNA protein.