Mediation of Proliferating Cell Nuclear Antigen (PCNA)-dependent DNA Replication through a Conserved p21Cip1-like PCNA-binding Motif Present in the Third Subunit of Human DNA Polymerase δ

Abstract

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 p21Cip1 . In accordance with this, a peptide containing the PCNA-binding domain of p21Cip1 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 Cip1 . 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 ␦.DNA replication requires the recruitment of multiple components during the S phase of the cell cycle. Of chief importance are DNA polymerases, of which three, ␣, ␦, and ⑀, have been shown by genetic studies in the yeast Saccharomyces cerevisiae to be essential for the replication of the eucaryotic genome (1, 2). Among these, DNA polymerase ␣-primase is the only enzyme that can start DNA synthesis de novo. It is thus ideally suited for the synthesis of short primers that serve to initiate leading strand synthesis at the replication origin and Okazaki fragments on the lagging strand (3, 4). Both DNA polymerases ␦ and ⑀ can elongate primers synthesized by DNA polymerase ␣ and appear to have overlapping or complementary functions (5). Although DNA polymerase ␦ alone can replicate both leading and lagging strands in vitro (5), mounting evidence suggests that DNA polymerase ⑀ may be implicated in DNA replication in ways that may not necessarily involve its DNA polymerase activity (6 -10).We have been interested in how DNA polymerase ␦ interacts with other factors to accomplish replication of the mammalian genome. Extensive research over a number of years has shown that DNA polymerase ␦ requires interactions with at least two factors, a toroidal-shaped homotrimeric protein known as proliferating cell nuclear antigen (PCNA) 1 and a heteropentameric complex termed replication factor C. In the presence of ATP, replication factor C interacts with the 3Ј primer-template terminus and recruits PCNA onto the DNA, creating a mobile platform that tethers DNA polymerase ␦ to the primer terminus. Once bound to PCNA, DNA polymerase ␦ acquires new dynamic properties, including high processivity, low nonpro...

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation

“…Our data suggest that cell cycle-dependent phosphorylation is a novel regulatory mechanism for Fen1 by directing cells to exit from S phase and demonstrate that Cdks enhance the end of the DNA replication by inactivating a protein involved in DNA replication fork progression. Cell cycle regulation of other DNA replication proteins such as DNA polymerase d (Wu et al, 1998;Ducoux et al, 2001), replication protein A (Dutta and Stillman, 1992), DNA polymerase a/primase (Voitenleitner et al, 1997;Schub et al, 2001) and DNA ligase I (Rossi et al, 1999) have been described. The modification of each protein upon phosphorylation by an active kinase in S phase leads to a negative effect on their function.…”

“…In mitosis, the nuclear lamina is disassembled by a direct Cdk1-Cyclin B phosphorylation (Nigg, 1995). DNA replication proteins such as large SV40 T antigen (McVey et al, 1993), DNA polymerase a/primase (Voitenleitner et al, 1997;Schub et al, 2001), DNA polymerase d (Wu et al, 1998;Ducoux et al, 2001), replication protein A (Dutta and Stillman, 1992) and DNA ligase I (Rossi et al, 1999) are all substrates for Cdk-Cyclin (reviewed in Henneke et al, 2003). In addition to Cdks, other protein-modifying factors such as protein phosphatase 2A (PP2A) and Cdc7-Dbf4 kinase (DDK) have essential functions in triggering S phase and initiating DNA replication (Lin et al, 1998;Johnston et al, 1999Johnston et al, , 2000.…”

Direct Interaction of p21 with p50, the Small Subunit of Human DNA Polymerase Delta

“…The small subunit, p50 (Fig. 5C), and the third subunit, p68, 33 of pol δ can also be phosphorylated in vitro by CDK2/cyclinA. The CDK2/cyclinA complex is associated with the pol δ complex even after a 0.4M NaCl wash of the anti-p125 immunoaffinity column and p50 antibody could readily immunoprecipitate cyclinA (Fig.…”

“…The latter could include several possibilities, i.e., the inhibition of pol δ by binding of p21 to PCNA within the replication complex, or by inhibition of phosphorylation of replication complex proteins by the CDK2/cyclinA kinase. The target substrates could include pol δ itself, since p50, p125 32 and p68 33 are potential substrates of CDK/cyclins.…”

“…32 p68/p66, the third subunit of pol δ is also a phosphoprotein in vivo and could be phosphorylated by CDKs/cyclins in vitro. 33 To assess if p50 can be phosphorylated in vivo, we transfected COS-7 cells with p50 cDNA in the pCI mammalian expression vector (Promega) with cMyc-His tags. After metabolic labeling with 32 Pi, the cells were immunoprecipitated with monoclonal antibody (17D2) against p50 (Figs.…”