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Vol. 10, Issue 7, 2119-2129, July 1999
Laboratory of Molecular Genetics, National Institute on Aging,
National Institutes of Health, Baltimore, Maryland 21224-6823
The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor
of cyclin-dependent kinases (CDKs). p21 can also block DNA replication
through its interaction with the proliferating cell nuclear antigen
(PCNA), which is an auxiliary factor for polymerase 
. PCNA is also
implicated in the repair resynthesis step of nucleotide excision repair
(NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this
controversy is of interest because it would 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 terminus) or the
PCNA binding domain (C terminus) of the protein. In the in vitro
studies, DNA repair synthesis was measured in extracts from normal
human fibroblasts using plasmids damaged by UV irradiation. In the in
vivo studies, we used intact and permeabilized cells. The results show
that the C terminus 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 vitro 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 DNA repair and replication, with
repair being much less sensitive to inhibition than replication. Our in
vivo results suggest that the inhibition occurs at the resynthesis step
of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding
of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and
the inhibition of DNA repair by the C terminus of p21 was relieved by
the addition of purified PCNA protein.
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