Mechanism of Inhibition of Proliferating Cell Nuclear Antigen-Dependent DNA Synthesis by the Cyclin-Dependent Kinase Inhibitor p21

Abstract

It is known that the direct binding of the cyclin-dependent kinase (Cdk) inhibitor p21, also called Cdk-interacting protein 1 (p21), to proliferating cell nuclear antigen (PCNA) results in the inhibition of PCNA-dependent DNA synthesis. We provide evidence that p21 first inhibits the replication factor C-catalyzed loading of PCNA onto DNA and second prevents the binding of DNA polymerase delta core to the PCNA clamp assembled on DNA. The second effect contributes most to the inhibition of pol delta holoenzyme activity. p21 primarily inhibited the DNA synthesis resulting from multiple reassembly of DNA polymerase delta holoenzyme. On the other hand, an ability of the PCNA clamp to translocate along double-stranded DNA was not affected by p21. These data were confirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibited clamp assembly nor prevented the loading of DNA polymerase delta core onto DNA. Our data suggest that p21 does not discriminate in vitro "repair" and "replication" DNA synthesis based on template length but does act preferentially on polymerization which encounters obstacles to progress.

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

“…PCNA is fundamental to nuclear DNA replication, acting as a sliding clamp that tethers the leading strand DNA polymerase d to its template, enhancing the enzyme's processivity (Prelich et al, 1987;Bravo et al, 1987;Krishna et al, 1994). In addition, PCNA is required for nucleotide excision repair (Shivji et al, 1992;Nichols and Sancar, 1992) and is involved in one pathway of base excision repair (Matsumoto et al, 1994).…”

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

“…A likely explanation is suggested by what is known about the e ects of p21 on PCNA and DNA polymerases. Although there is a modest e ect of p21 on the ability of RFC to load the PCNA toroid onto DNA, the principal consequence of p21 bound to PCNA is that it blocks association of polymerase with the PCNA clamp, thereby preventing synthesis (Podust et al, 1995a). This suggests the following scenario.…”

“…Synthesis of longer tracts of DNA as in DNA replication will be less straightforward because DNA polymerase d undergoes multiple cycles of dissociation and reassociation with PCNA. Any reassociation of p21 with PCNA during this polymerase dissociationreassociation cycle will again disrupt replicative synthesis (Podust et al, 1995a).…”

“…PCNA forms a trimeric toroid structure that serves as a sliding clamp for DNA polymerase delta (pol d) and DNA polymerase epsilon (pol e) holoenzymes on DNA. This binding of p21 to PCNA can inhibit DNA synthesis by blocking association of DNA polymerase with PCNA (Podust et al, 1995a). Binding of p21 to PCNA can also interfere with replication by preventing binding of the FEN1 5'-3' replicative exonuclease to the replicative complex (Chen et al, 1996a;Warbrick et al, 1997).…”

hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress

“…Phosphorylation of p53 on Ser15 or Ser20 stabilizes p53 against proteasomal degradation (Brooks and Gu, 2003). Stabilized p53 serves as a transcription factor and stimulates expression of the cyclin-dependent kinase inhibitor p21 which activates cell cycle checkpoints by inhibiting cyclin/CDK complexes and PCNA, and also promotes cell survival in response to oxidative stress (elDeiry et al, 1993;Podust et al, 1995;O'Reilly et al, 2001). Tight regulation of the p53 pathway in response to genotoxic stress is critical in ensuring the optimal outcome since it controls whether cells undergo programmed cell death or survive in response to DNA damage (O'Connor, 1997).…”