Abstract
p21, a p53-induced gene product that blocks cell cycle progression at the G1 phase, interacts with both cyclin-dependent kinases and proliferating cell nuclear antigen (PCNA). PCNA functions as a processivity factor for DNA polymerases delta and epsilon and is required for both DNA replication and nucleotide excision repair. Previous studies have shown that p21 inhibits simian virus 40 (SV40) DNA replication in HeLa cell extracts by interacting with PCNA. In this report we show that p21 blocks nucleotide excision repair of DNA that has been damaged by either ultraviolet radiation or alkylating agents, and that this inhibition can be reversed following addition of PCNA. We have determined that p21 is more effective in blocking DNA resynthesis than in inhibiting the excision step. We further show that a peptide derived from the carboxyl terminus of p21, which specifically interacts with PCNA, inhibits polymerase delta-catalyzed elongation of DNA chains almost stoichiometrically relative to the concentration of PCNA. When added at higher levels, this peptide also blocks both SV40 DNA replication and nucleotide excision repair in HeLa cell extracts. These results indicate that p21 interferes with the function of PCNA in both in vitro DNA replication and nucleotide excision repair.
Highlights
We further show that a peptide derived from the carboxyl terminus of p21,which interacts with proliferating cell nuclear antigen (PCNA), inhibits polymerase δ-catalyzed elongation of DNA chains almost stoichiometrically relative to the concentration of PCNA
In order to assess the influence of p21 on the PCNA-dependent repair reaction, we initially examined the efficiency of the repair synthesis reaction as a function of both time and the concentration of HeLa extract added for the repair of UVdamaged DNA
Warbrick et al (1995) have shown that a peptide derived from the carboxyl domain of p21 interacts with PCNA and inhibits simian virus 40 (SV40) DNA replication catalyzed by crude cytosolic extracts
Summary
This substrate were purchased from Operon Technologies, Inc. or Midland Certified Reagent Co., T4 polynucleotide kinase and T4 DNA. When the influence of p21 protein or the p21 carboxyl-terminal peptide on repair synthesis was examined, reactions containing the inhibitors (diluted with a solution containing 50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, and 1 mM EDTA) were preincubated for 15 min at 30 °C with HeLa CFE (50 μg). This was followed by the addition of the other components, and the DNA was processed as described for the basic repair synthesis reaction. DNA Replication Assays —SV40 DNA replication and the elongation of singly primed M13 DNA were carried out as described previously (Wobbe et al, 1985; Flores-Rozas et al, 1994)
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