Mediation of Proliferating Cell Nuclear Antigen (PCNA)-dependent DNA Replication through a Conserved p21-like PCNA-binding Motif Present in the Third Subunit of Human DNA Polymerase *

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase has not been clearly defined. We show that the third subunit of human DNA polymerase , p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase activity and for binding of the cell cycle inhibitor p21. In accordance with this, a peptide containing the PCNA-binding domain of p21 inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase . Furthermore, pull-down assays showed that DNA polymerase requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21. Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase .