(The) Cdc7/Dbf4-mediated phosphorylation of Dna2 is required for S-phase progression in a Mec1-dependent manner

Abstract

Dna2 is a nuclease/helicase that is involved in multiple DNA transactions including Okazaki fragment processing, double-strand break resection, and S-phase checkpoint activation. Since Dna2 plays a key role in many cellular processes, major efforts have been concentrated to elucidate its signaling network, mainly by identification of Dna2 interacting partners. In this study, we found that Cdc7/Dbf4, an essential kinase complex that plays crucial roles in the initiation of DNA replication and cell cycle progression in eukaryotes, physically interacts with Dna2 in a yeast two-hybrid screening. In addition, purified recombinant Dna2 and Cdc7/Dbf4 were able to bind each other in ELISA assays. We also present evidence that the Cdc7/Dbf4 kinase complex efficiently phosphorylated Dna2 both in vitro and in vivo. The phosphorylation of Dna2 by Cdc7/Dbf4, which occurred primarily at the N-terminal region of Dna2, resulted in decreased binding of Dna2 to flap DNA substrates, thereby lowering its endonuclease activity in vitro. Moreover, we show that Dna2, when expressed in a multi-copy plasmid, rescued the temperature-sensitive growth defect of the cdc7-4 and dbf4-1 mutants and that overexpression of Dna2 allowed cell-cycle arrested dbf4-1 mutant cells to efficiently progress into S phase in a manner dependent on the Mec1 kinase. This required the functional interactions between Dna2 and Mec1 since the Trp128 and Tyr130 residues of Dna2 were essential for the activation of the Mec1 kinase. Our results demonstrate for the first time that there is a functional link between Dna2 and the cell cycle kinase complex Cdc7/Dbf4. They interacted both genetically and physically, and these interactions are important for the timely progression of S-phase in a Mec1-dependent manner.