Resistance of human nucleotide excision repair synthesis in vitro to p21(Cdn1)

The p21(Cdn1) protein (cipl/waf1/sdi1) plays an important role as an inhibi tor of mammalian cell proliferation in response to DNA damage. By interacti ng with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues , even though this pathway is PCNA-dependent. We investigated features of p 21-resistant NER using human cell extracts. A direct end-labelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, filling of the similar to 30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap filling was uncoupled from incision. Binding p21 to PCNA could al so inhibit filling of model similar to 30 nt gaps by both purified DNA poly merases delta and epsilon. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved wit h time. This incubation gives p21 the opportunity to associate with other t argets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap filling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an efficient inhibitor of NER synthesis in cell extracts.