A Fission Yeast Gene,him1⁺/dfp1⁺, Encoding a Regulatory Subunit for Hsk1 Kinase, Plays Essential Roles in S-Phase Initiation as Well as in S-Phase Checkpoint Control and Recovery from DNA Damage

Abstract

Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G₁/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1⁺ from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1⁺ is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G₁. The protein level is low at START in G₁, increases at the G₁/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G₁/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1⁺ is identical to rad35⁺. Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

ACKNOWLEDGMENTS

We are grateful to Takahisa Hachiya and Katsuyuki Tamai (MBL) for help in generation of antibodies against Him1 protein. We thank Masashi Uchiyama for help in synchronization experiments. We are also grateful to Koichi Tanaka and Hiroto Okayama for the gift of cdc10-V50 and cds1⁻ strains and to Paul Nurse for the gift of Orp1HA-tagged strain. We thank Hiromi Iiyama for excellent technical assistance and Keiji Tanaka, Asako Sawano, and Masafumi Shibuya for advice on handling insect cells and for permitting us access to their laboratory facility. We also thank Noriko Sato for critical reading of the manuscript and our colleagues in the laboratory for valuable discussions and comments.