Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.
The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, 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 delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta 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 delta.
Ducoux
M
M
Institut Curie, UMR 2027 du CNRS, Génotoxicologie et Cycle Cellulaire, Bâtiment 110, Centre Universitaire, 91405 Orsay Cedex, France.
Urbach
S
S
Baldacci
G
G
Hübscher
U
U
Koundrioukoff
S
S
Christensen
J
J
Hughes
P
P
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D011994:Recombinant Proteins; C435720:POLD3 protein, human; D004258:DNA Polymerase III