The yeast RAPl protein is a sequence-specific DNA-binding protein that functions as both a repressor and an activator of transcription. RAPl is also involved in the regulation of telomere structure, where its binding sites are found within the terminal poly(Ci_3A) sequences. Previous studies have indicated that the regulatory function of RAPl is determined by the context of its binding site and, presumably, its interactions with other factors. Using the two-hybrid system, a genetic screen for the identification of protein-protein interactions, we have isolated a gene encoding a RAPl-interacting factor (RIFl). Strains carrying gene disruptions of RIFl grow normally but are defective in transcriptional silencing and telomere length regulation, two phenotypes strikingly similar to those of silencing-defective rapl" mutants. Furthermore, hybrid proteins containing rapl^ missense mutations are defective in an interaction with RIFl in the two-hybrid system. Taken together, these data support the idea that the rapT phenotypes are attributable to a failure to recruit RIFl to silencers and telomeres and suggest that RIFl is a cofactor or mediator for RAPl in the establishment of a repressed chromatin state at these loci. By use of the two-hybrid system, we have isolated a mutation in RIFl that partially restores the interaction with rapl** mutant proteins.

The Rif1 protein, originally identified as a telomere-binding factor in yeast, has recently been implicated in DNA replication control from yeast to metazoans. Here, we show that budding yeast Rif1 protein inhibits activation of prereplication complexes (pre-RCs). This inhibitory function requires two N-terminal motifs, RVxF and SILK, associated with recruitment of PP1 phosphatase (Glc7). In G1 phase, we show both that Glc7 interacts with Rif1 in an RVxF/SILK-dependent manner and that two proteins implicated in pre-RC activation, Mcm4 and Sld3, display increased Dbf4-dependent kinase (DDK) phosphorylation in rif1 mutants. Rif1 also interacts with Dbf4 in yeast two-hybrid assays, further implicating this protein in direct modulation of pre-RC activation through the DDK. Finally, we demonstrate Rif1 RVxF/SILK motif-dependent recruitment of Glc7 to telomeres and earlier replication of these regions in cells where the motifs are mutated. Our data thus link Rif1 to negative regulation of replication origin firing through recruitment of the Glc7 phosphatase.

Cdc7p is a protein kinase that is required for G 1 ͞S transition and initiation of DNA replication in Saccharomyces cerevisiae. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. We report here the characterization in S. cerevisiae of a recessive mutation in a member of the MCM family, MCM5͞CDC46, which bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, our studies suggest that Cdc7p is required late in G 1 because in its absence the Mcm5p͞Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p͞Cdc46p may have both positive and negative effects on the ability of cell to initiate replication.

Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. We have determined that the levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G 2 /M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. We report that Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). We have determined that Cdc5p-associated kinase activity is restricted to G 2 /M and that this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis.

DNA replication initiates from specific chromosomal sites called origins, and in the budding yeast Saccharomyces cerevisiae these sites are occupied by the origin recognition complex (ORC). Dbf4p is proposed to play a role in targeting the G 1 /S kinase Cdc7p to initiation complexes late in G 1 . We report that Dbf4p may also recruit Cdc5p to origin complexes. Cdc5p is a member of the Polo family of kinases that is required for the completion of mitosis. Cdc5p and Cdc7p each interact with a distinct domain of Dbf4p. cdc5-1 mutants have a plasmid maintenance defect that can be suppressed by the addition of multiple origins. cdc5-1 orc2-1 double mutants are synthetically lethal. Levels of Cdc5p were found to be cell cycle regulated and peaked in G 2 /M. These results suggest a role for Cdc5p and possibly Polo-like kinases at origin complexes.In order to maintain the integrity of its genome, a eukaryotic cell must not only replicate its DNA accurately but also limit replication to once per cell cycle. Initiation of DNA replication requires both cis-and trans-acting factors (58). The cis-acting element is a DNA sequence called an origin. In Saccharomyces cerevisiae, a DNA sequence which is capable of supporting autonomous replication both on plasmids and in a chromosomal context has been identified (8,46). Linker substitution analysis of three such autonomously replicating sequence (ARS) elements, ARS1, ARS305, and ARS307, shows that the ARS consists of several DNA elements, including an essential 11-bp consensus sequence (ACS) and a B region with at least two elements, B1 and B2 (8, 45, 52, 60). A complex of six proteins, the origin recognition complex (ORC), serves as a trans-acting factor. ORC binds to the B1 and ACS elements in a sequence-specific, ATP-dependent manner (3,4,16,53,55).Analysis of replication origins via in vivo DNase I footprinting reveals that during the cell cycle, budding yeast origins are bound by at least two distinct complexes, termed the prereplicative and postreplicative complexes (17). The postreplicative complex, present from S phase through late M phase, resembles the in vitro footprint produced with purified ORC (17). A transition to the prereplicative complex occurs late in M and persists until late G 1 /early S phase. The prereplicative complex displays an extended region of nuclease protection which encompasses the ORC footprint. The binding of ORC to origins appears to be fundamental, but not sufficient, for establishing a competent initiation complex. ORC appears to be bound to origins throughout the cell cycle (55), and therefore additional trans-acting factors must play a role in establishing replicationcompetent origins either as prereplication factors or as modifiers of origin-bound ORC.The formation of the prereplicative complex is proposed to be the first step in priming the origin for initiation of DNA replication (17). The final step is the activation of the prereplicative complexes, leading to the unwinding of the origin sequences and the eventual recruitment of the DNA ...