Abstract
In eukaryotes, processive DNA synthesis catalyzed by DNA polymerases delta and epsilon (pol delta and epsilon) requires the proliferating cell nuclear antigen (PCNA). It has recently been shown that in humans (h), the PCNA function, required for both DNA replication and nucleotide excision repair, can be inactivated by p21(CIP1) due to a specific interaction between hPCNA and the carboxyl terminus of p21(CIP1). In this report, we show that Saccharomyces cerevisiae (S. cerevisiae) PCNA-dependent pol delta-catalyzed DNA synthesis was inhibited less efficiently than the human system by the intact p21(CIP1) protein and was unaffected by the p21(CIP1) carboxyl-terminal peptide (codons 139-160). This species-specific response of PCNA to p21(CIP1)-mediated inhibition of DNA synthesis results from a marked difference in the ability of h and S. cerevisiae PCNA to interact with p21(CIP1). As shown by binding studies using the surface plasmon resonance technique, hPCNA binds both full-length p21(CIP1) and the p21(CIP1) peptide-(139-160) stoichiometrically with a similar affinity (KD approximately 2.5 nM) while S. cerevisiae PCNA binds p21(CIP1) with approximately 10-fold less affinity and does not interact with the p21(CIP1) peptide-(139-160).
Highlights
** American Cancer Society Professor. 1 The abbreviations used are: pol, polymerase; gp, gene product; h, human; HSSB, human single-stranded DNA binding protein; PCNA, proliferating cell nuclear antigen; RFC, replication
Reactions containing S. cerevisiae pol ␦ and S. cerevisiae RFC with hPCNA were inhibited by the p21CIP1 peptide-(139 –160), whereas reactions containing h or S. cerevisiae pol ␦, S. cerevisiae RFC, and S. cerevisiae PCNA were unaffected by the peptide
Real time interaction analysis presented here demonstrated that the affinity of the p21CIP1 for hPCNA was ϳ15-fold greater than its affinity for S. cerevisiae PCNA
Summary
** American Cancer Society Professor. 1 The abbreviations used are: pol, polymerase; gp, gene product; h, human; HSSB, human single-stranded DNA binding protein ( called RP-A); PCNA, proliferating cell nuclear antigen; RFC, replication. PCNA has been shown to be the target of a number of factors that control cell growth One such factor, p21CIP1, is a checkpoint protein that acts as an antimitogenic signal by binding to and inhibiting cyclin-dependent kinases as well as by binding to PCNA and inhibiting in vitro PCNA-dependent DNA replication [22,23,24,25,26,27,28,29]. We have examined the species specificity involved in the pol ␦ holoenzyme system using enzymes isolated from S. cerevisiae and HeLa cells and have shown that a chemically synthesized peptide that spans amino acid residues 139 –160 of p21CIP1 inhibits reactions dependent on human PCNA.
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