Cell Cycle-dependent Phosphorylation of the Large Subunit of Replication Factor C (RF-C) Leads to Its Dissociation from the RF-C Complex

Abstract

The five subunit replication factor C (RF-C) complex plays a critical role in DNA elongation. We find that the large subunit of RF-C (RF-Cp145) is phosphorylated in vivo whereas the smaller RF-C subunits are not phosphorylated. The phosphorylation of endogenous RFCp145 is modulated in a cell cycle-dependent manner. Phosphorylation is maximal in G 2 /M and is inhibited by an inhibitor of cyclin-dependent kinases. Phosphorylation of purified recombinant RF-C complex in vitro reveals that RF-Cp145 is preferentially phosphorylated by cdc2-cyclin B but not by cdk2-cyclin A or cdk2-cyclin E. In vitro phosphorylation of RF-C complex by cdc2-cyclin B kinases leads to dissociation of phosphorylated RFCp145 from the RF-C complex. Using different approaches we demonstrate that phosphorylated RFCp145 is indeed dissociated from RF-Cp40 and RF-Cp37 in vivo. These results suggest that destabilization of the RF-C complex by CDKs may inactivate the RF-C complex at the end of S phase.Cell cycle progression is regulated by distinct cyclin-dependent kinases (CDKs), 1 which activate at different times in the cell cycle. In mammalian cells, cyclin E-dependent kinase is activated at the G 1 to S phase transition, after the D-type cyclins but prior to A-type cyclins. The timing of activation of cyclin A kinase activity coincides with the onset of DNA synthesis (1). Cdc2-cyclin B kinase activation at the G 2 /M transition is required for cells to enter mitosis. Replication of DNA is a highly regulated process, which occurs during the S phase of the cell cycle. The DNA polymerase ␣-primase complex allows the generation of RNA-DNA primers for initiation of leading strand synthesis and synthesis of each Okazaki fragment during lagging strand replication. The polymerase switch from DNA polymerase ␣ to DNA polymerase ␦ then catalyzes the replication of the leading strand and for completion of the lagging strand. The replication factor C (RF-C) complex and PCNA are essential for processive DNA synthesis. RF-C loads PCNA onto DNA in an ATP-dependent process (2, 3). The PCNA clamp then recruits DNA polymerase ␦ for processive DNA synthesis (4).RF-C is a five subunit heterodimer, and all five subunits of RF-C from human and yeast have been cloned (5-13). RF-C activity can be reconstituted in vitro when all five subunits are expressed as recombinant proteins (14 -16).The Escherichia coli clamp loader, the ␥ complex, like the yeast and human RF-C complexes is comprised of five polypeptides. The DNA-dependent ATPase activity of the clamp in the ␥ complex is provided by three copies of the ␥ polypeptide whereas human (and yeast) utilize three different polypeptide chains hRF-Cp37 (yRF-C2), hRF-Cp36 (yRF-C3), and hRFCp40 (yRF-C4). The ␦ subunit (homologous to yRF-C1 in yeast and hRF-Cp145 in man) binds the clamp and has conserved hydrophobic residues required for clamp loading. Structural studies of the ␥ complex: ␦Ј-␥1-␥2-␥3-␦ reveals that the Cterminal domains of ␦Ј, ␥, and ␦ form a helical scaffold (circular collar) and the N-terminal ends appear ...

The Second Subunit of the Replication Factor C complex (RFC40) and the Regulatory Subunit (RI?) of Protein Kinase A form a Protein Complex Promoting Cell Survival

“…Coimmunoprecipitation experiments using antibodies against RFC140 (for the RFC1-5 complex) and RFC37 (for the RFC2-5 complex), respectively, were performed. As previously demonstrated, 17 RFC140 coimmunoprecipitated with RFC40 (Fig. 5A, left panel) but not with RIα (Fig.…”

Identification of RFC5 as a novel potential prognostic biomarker in lung cancer through bioinformatics analysis

“…A study has indicated that RFC catalyzed the formation of a cyclic structure of PCNA around the primers in an ATP-dependent manner ( 35 ). Munshi et al ( 36 ) observed that cyclin-dependent kinases reduced the stability of RFC, inactivating it in the S phase to regulate DNA replication ( 36 ). However despite being an important component of the RFC, the role of RFC5 in lung cancer remains unknown.…”

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

“…40 Several major replication proteins such as DNA polymerase alpha, replication factor C, and Fen 1 are phosphorylated by CDK/ cyclins both in vitro and in vivo. [41][42][43][44][45] Our earlier work showed that pol δ catalytic subunit, p125, 32 is a potential substrate of CDKs/ cyclins in vivo. The small subunit, p50 (Fig.…”