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Rif1 is a key factor for spatiotemporal regulation of DNA replication. Rif1 suppresses origin firing in the mid-late replication domains by generating replication-suppressive chromatin architecture and by recruiting a protein phosphatase., Rif1 is a key factor for spatiotemporal regulation of DNA replication. Rif1 suppresses origin firing in the mid-late replication domains by generating replication-suppressive chromatin architecture and by recruiting a protein phosphatase. In fission yeast, the function of Hsk1, a kinase important for origin firing, can be bypassed by rif1Δ due to the loss of origin suppression. Rif1 specifically binds to G-quadruplex (G4) in vitro. Here, we show both conserved N-terminal HEAT repeats and C-terminal nonconserved segments are required for origin suppression. The N-terminal 444 amino acids and the C-terminal 229 amino acids can each mediate specific G4 binding, although high-affinity G4 binding requires the presence of both N- and C-terminal segments. The C-terminal 91 amino acids, although not able to bind to G4, can form a multimer. Furthermore, genetic screening led to identification of two classes of rif1 point mutations that can bypass Hsk1, one that fails to bind to chromatin and one that binds to chromatin. These results illustrate functional domains of Rif1 and indicate importance of both the N-terminal HEAT repeat segment and C-terminal G4 binding/oligomerization domain as well as other functionally unassigned segments of Rif1 in regulation of origin firing.

Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of mrc1 checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 bypass segment) on Mrc1. An ΔHBS mutant does not activate late/dormant origin firing in the presence of hydroxyurea but causes the precocious and enhanced activation of weak early-firing origins during normal S-phase progression and bypasses the requirement for Hsk1 for growth. This may be caused by the disruption of intramolecular binding between HBS and NTHBS (N-terminal target of HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting the intramolecular interaction. We propose that Mrc1 exerts a “brake” on initiation (through intramolecular interactions) and that this brake can be released (upon the loss of intramolecular interactions) by either the Hsk1-mediated phosphorylation of Mrc1 or the deletion of HBS (or a phosphomimic mutation of putative Hsk1 target serine/threonine), which can bypass the function of Hsk1 for growth. The brake mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.

Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G(1)/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 G(1). The protein level is low at START 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 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.

A novel human protein, ASK (activator of S phase kinase), was identified on the basis of its ability to bind to human Cdc7-related kinase (huCdc7). ASK forms an active kinase complex with huCdc7 that is capable of phosphorylating MCM2 protein. ASK appears to be the major activator of huCdc7, since immunodepletion of ASK protein from the extract is accompanied by the loss of huCdc7-dependent kinase activity. Expression of ASK is regulated by growth factor stimulation, and levels oscillate through the cell cycle, reaching a peak during S phase. Concomitantly, the huCdc7-dependent kinase activity significantly increases when cells are in S phase. Furthermore, we have demonstrated that ASK serves an essential function for entry into S phase by showing that microinjection of ASK-specific antibodies into mammalian cells inhibited DNA replication. Our data show that ASK is a novel cyclin-like regulatory subunit of the huCdc7 kinase complex and that it plays a pivotal role in G1/S transition in mammalian cells.

How early- and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here, we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in the fission yeast Schizosaccharomyces pombe. In mrc1Δ cells (and in swi1Δ cells to some extent), efficiency of firing is stimulated, and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1Δ cells. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that prefiring binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence of this protein.