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

Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required fo r G(1)/S transition, and its related kinases are present in fission yeast a s well as in higher eukaryotes, including humans. Kinase activity of Cdc7 p rotein depends on the regulatory subunit, Dbf4, which also interacts with r eplication origins. We have identified him1(+) from two-hybrid screening,vi th Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encod es a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identi fied as an associated molecule of Hsk1, binds to Hsk1 and stimulates its ki nase 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 reg ulated, increasing at middle M to late G(1). The protein level is low at ST ART in G(1), increases at the G(1)/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G(1)/S thr ough 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 substit ution of three serine and threonine residues present in the same motif resu lted in a defect in checkpoint regulation normally induced by hydroxyurea t reatment. The alanine mutant also showed growth retardation after UV irradi ation and the addition of methylmethane sulfonate. In keeping with this res ult, 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 i n addition to its predicted essential function in S-phase initiation.