Abstract
Clf1p is an essential, highly conserved protein in S. cerevisiae that has been implicated in pre-mRNA splicing. Clf1p's ortholog in Drosophila, Crn, is required for normal cell proliferation. Cells depleted of Clf1p arrest primarily with large buds, a single nucleus, a 2C DNA content, and a short, intact mitotic spindle. We isolated temperature-sensitive clf1 mutants that exhibit similar mitotic defects when released to the restrictive temperature from an early S-phase block. While these mutants also accumulate unspliced pre-mRNA at the restrictive temperature, the mitotic arrest does not appear to result from a failure to splice tubulin pre-mRNA. Moreover, the same mutants exhibit a delayed entry into S phase when released to the restrictive temperature from a G1 phase block. This delay could not be suppressed by disruption of the S-phase CDK inhibitor SIC1, suggesting that Clf1p is involved in DNA replication. Consistent with this possibility, we find that Clf1p (but not the mutant clf1p) interacts with the DNA replication initiation protein Orc2p in two-hybrid and co-immunoprecipitation assays, that Clf1p preferentially associates with origins of DNA replication, and that this association is Orc2p dependent. These observations suggest that Clf1p plays a direct role in the initiation of DNA replication.