Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

The ring-shaped complex PCNA coordinates DNA replication, encircling DNA to act as a polymerase clamp and a sliding platform to recruit other replication proteins. PCNA is loaded onto DNA by replication factor C, but it has been unknown how PCNA is removed from DNA when Okazaki fragments are completed or the replication fork terminates. Here we show that the Elg1 replication factor C-like complex (Elg1-RLC) functions in PCNA unloading. Using an improved degron system we show that without Elg1, PCNA accumulates on Saccharomyces cerevisiae chromatin during replication. The accumulated PCNA can be removed from chromatin in vivo by switching on Elg1 expression. We find moreover that treating chromatin with purified Elg1-RLC causes PCNA unloading in vitro. Our results demonstrate that Elg1-RLC functions in unloading of both unmodified and SUMOylated PCNA during DNA replication, while the genome instability of an elg1Δ mutant suggests timely PCNA unloading is critical for chromosome maintenance.

We discovered that sequences essential for replication origin function are frequently conserved in sensu stricto Saccharomyces species. Here we use analysis of phylogenetic conservation to identify replication origin sequences throughout the Saccharomyces cerevisiae genome at base pair resolution. Origin activity was confirmed for each of 228 predicted sites-representing 86% of apparent origin regions. This is the first study to determine the genome-wide location of replication origins at a resolution sufficient to identify the sequence elements bound by replication proteins. Our results demonstrate that phylogenetic conservation can be used to identify the origin sequences responsible for replicating a eukaryotic genome.Supplemental material is available at http://www.genesdev.org.

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

Replication of eukaryotic chromosomes initiates at multiple sites called replication origins. Replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where several complementary studies have mapped their locations genome-wide. We have collated these datasets, taking account of the resolution of each study, to generate a single list of distinct origin sites. OriDB provides a web-based catalogue of these confirmed and predicted S.cerevisiae DNA replication origin sites. Each proposed or confirmed origin site appears as a record in OriDB, with each record comprising seven pages. These pages provide, in text and graphical formats, the following information: genomic location and chromosome context of the origin site; time of origin replication; DNA sequence of proposed or experimentally confirmed origin elements; free energy required to open the DNA duplex (stress-induced DNA duplex destabilization or SIDD); and phylogenetic conservation of sequence elements. In addition, OriDB encourages community submission of additional information for each origin site through a User Notes facility. Origin sites are linked to several external resources, including the Saccharomyces Genome Database (SGD) and relevant publications at PubMed. Finally, a Chromosome Viewer utility allows users to interactively generate graphical representations of DNA replication data genome-wide. OriDB is available at .

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation‐mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N‐terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1‐PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1‐PP1 protects the origin‐binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1‐depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1‐targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.

Abstract. The 42-kD component of the S. cerevisiae spindle pole body (SPB) localizes to the electron-dense central plaque of the SPB (Rout, M.P., and J.V. Kilmartin. 1991. Cold Spring Harbor Symp. Quant. Biol. 56:687--691). We have cloned the corresponding gene SPC42 (spindle pole component) and show that it is essential. Seven temperature-sensitive (ts) mutants in SPC42 were prepared by error-prone PCR. We found that a change to a proline residue in a potential coiledcoil region of Spc42p was responsible for the ts phenotype in at least three alleles, suggesting that formation of the coiled-coil is essential for normal function. The mutant cells showed a phenotype of predominantly single or bilobed SPBs often with an accumulation of unstructured electron-dense material associated with the bridge structure adjacent to the SPB. This phenotype suggests a defect in SPB duplication. This was confirmed by examining synchronized mutant cells that lose viability when SPB duplication is attempted. Spc42p is a phosphoprotein which shows some cell cycle-regulated phosphorylation. Overexpression of Spc42p causes the formation of a disc-or dome-shaped polymer composed of phosphorylated Spc42p, which is attached to the central plaque and associated with the outer nuclear membrane. Taken together, these data suggest that Spc42p forms a polymeric layer at the periphery of the SPB central plaque which has an essential function during SPB duplication and may facilitate attachment of the SPB to the nuclear membrane.T hE spindle pole body (SPB) 1 of Saccharomyces cerevisiae is the functional equivalent of the centrosome in the yeast cell. It is a complex cylindrical multi-layered structure lying in the nuclear envelope. The central layer or plaque lies in the plane of the nuclear membrane. On the cytoplasmic side is the outer plaque that organizes cytoplasmic microtubules, while nuclear or spindle microtubules are organized from a more illdefined inner plaque (Byers, 1981a;Rose et al., 1993;. Like the centrosome, the SPB is duplicated during G1/S. The daughter SPB assembles on the satellite structure, which is cytoplasmic and attached to the mother SPB by a short electron-dense segment of the nuclear membrane called the half-bridge (Byers and Goetsch, 1975;Winey et al., 1991a). During or after duplication the daughter SPB is inserted into the nuclear envelope. Both SPBs remain connected by the bridge until this structure is somehow partitioned between the two SPBs as they migrate to opposite positions on the nuclear envelope and form the mitotic spindle (Byers and Goetsch, 1974).