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.

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.

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase ⑀. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase ⑀ that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3-to 5-exonuclease-deficient mutant of DNA polymerase ␦ in a haploid cell. These results suggest that the catalytic activity of DNA polymerase ⑀ participates in the same pathway as DNA polymerase ␦, and this is consistent with the observation that DNA polymerases ␦ and ⑀ colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.Saccharomyces cerevisiae has three DNA polymerases (pol␣, -␦, and -⑀) 1 that are required for cell growth, chromosomal DNA replication (1), and DNA double-strand break repair (2). pol␣ consists of four subunits (Pol1p (Cdc17p), Pol10p, Pri1p, and Pri2p) and is primarily involved in the initiation of DNA replication and priming of Okazaki fragments. pol␦ and -⑀ are required during synthesis of the leading and lagging strands at the replication fork, binding at/or near replication origins, and moving along DNA with the replication fork (3, 4). The precise roles of pol␦ and pol⑀ during leading and lagging strand synthesis have yet not been defined; however, genetic and biochemical evidence suggests that lagging strand synthesis is carried out by pol␣ and pol␦ (5, 6). Nevertheless, simian virus 40 DNA replication only requires pol␣ and pol␦ (5).S. cerevisiae pol␦ contains the three subunits Pol3 (Cdc2), Hys2 (Pol31) (7), and Pol32 (8), which are homologues of Schizosaccharomyces pombe Pol3, Cdc1, and Cdc27, respectively. S. pombe pol␦ contains one additional subunit, Cmt1 (9). Purified yeast pol␦ requires accessory factors including PCNA and the RF-C to catalyze processive DNA synthesis; this suggests that pol␦ may be the leading strand DNA polymerase (5, 10). pol␦ has a 3Ј-to 5Ј-exonuclease, which acts as a proofreading/editing polymerase during DNA synthesis (11, 12).S. cerevisiae pol⑀ is also a multisubunit complex consisting of Pol2p, Dpb2p, Dpb3p, and Dpb4p (13,14). pol⑀ requires PCNA and RF-C complex to catalyze processive DNA synthesis on singly primed single-stranded viral DNA, although pol⑀ is a highly processive enzyme (13,15,16). Pol2p is the catalytic subunit of pol⑀, and it is encoded by the POL2 gene (17), which is essential in yeast. pol⑀ is a class B polymerase, characterized by six conserved domains (I-VI) in the N-terminal ha...