MECHANISM OF INHIBITION OF PROLIFERATING CELL NUCLEAR ANTIGEN-DEPENDENT DNA-SYNTHESIS BY THE CYCLIN-DEPENDENT KINASE INHIBITOR P2L

It is known that the direct binding of the cyclin-dependent kinase (Cd k) inhibitor p21, also called Cdk-interacting protein 1 (p21), to prol iferating cell nuclear antigen (PCNA) results in the inhibition of PCN A-dependent DNA synthesis. We provide evidence that p21 first inhibits the replication factor C-catalyzed loading of PCNA onto DNA and secon d 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 syn thesis resulting from multiple reassembly of DNA polymerase delta holo enzyme. On the other hand, an ability of the PCNA clamp to translocate along double-stranded DNA was not affected by p21. These data were co nfirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibited clamp assembly nor prevented the loading of DNA pol ymerase delta core onto DNA. Our data suggest that p21 does not discri minate in vitro ''repair'' and ''replication'' DNA synthesis based on template length but does act preferentially on polymerization which en counters obstacles to progress.