We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.DNA replication is a fundamental process of all dividing cells. During the eukaryotic cell cycle, DNA replication occurs exactly once and is initiated only upon the completion of mitosis. Strict regulation of timing appears to be mediated through the coordinated activities of numerous proteins. Due in large part to studies of viral DNA replication, the enzymatic activities at the replication fork were elucidated many years ago (33). In contrast, trans-acting factors which regulate the initiation of DNA replication have been described only recently. In an effort to gain a comprehensive understanding of the factors involved in this essential process, we have sought to identify gene products that influence the initiation of DNA replication.Saccharomyces cerevisiae provides an excellent eukaryotic model for identifying proteins involved in DNA replication. Many replication initiation factors currently under investigation are conserved in mammalian cells and were found initially in yeast. Members of the origin recognition complex (ORC) (20,22) were originally identified biochemically through their binding to the consensus sequence of autonomously replicating sequences (ARSs), which function as DNA replication origins (2). Our screen for yeast minichromosome maintenance (mcm) mutants (39) has also been fruitful in identifying replication initiation factors, such as those of the MCM2-7 family (9, 29, 52). The MCM2-7 proteins are a family of six conserved proteins that are ubiquitous in eukaryotes. Their essential role in the initiation of DNA synthesis not only has been demonstrated by in vivo studies in a number of organisms, including S. cerevisiae (55), Schizosaccharomyces pombe (40), and Drosophila melanogaster (51), but also is supported by in vitro studies in Xenopus laevis (8,34,38). The MCM2-7 proteins interact with one another, and possibly other proteins, to form large complexes (35). Despite their structural similarity, each of these proteins is indispensable for ...

MCM2 and MCM3 are two genetically interacting and structurally related proteins essential for growth inSaccharomyces cerevisiae. Mutants defective in these proteins affect the stability of minichromosomes in general, but the severity of the defect is dependent on the autonomously replicating sequence (ARS) that drives the replication of that plasmid. In this paper we show by two-dimensional gel electrophoresis that the initiation of DNA synthesis at chromosomal replication origins is also reduced in frequency in these mutants. We show further that the nuclear and subnuclear localizations of the MCM2 and MCM3 proteins are temporally regulated with respect to the cell cycle. These proteins enter the nucleus at the end of mitosis, persist there throughout G~ phase, and disappear from it at the beginning of S phase. Once inside the nucleus, a fraction of the MCM2 and MCM3 proteins becomes tightly associated with DNA. The association of MCM2 and MCM3 with chromatin presumably leads to the initiation of DNA synthesis, and their subsequent disappearance from the nucleus presumably prevents reinitiation of DNA synthesis at replication origins. This temporally and spatially restricted localization of MCM2 and MCM3 in the nucleus may serve to ensure that DNA replication occurs once and only once per cell cycle.[Key Words: MCM2; MCM3; S. cerevisiae; nuclear localization; initiation of DNA synthesis; DNA replication; licensing factors] Received July 13, 1993; revised version accepted August 24, 1993.Regulation of DNA replication in eukaryotes is dovetailed with stages of the cell cycle. In each cell cycle, (1) replication occurs only during S phase, (2) chromosomal re-replication is prevented, (3) mitosis is delayed until replication is completed, and (4) the re-replication block is removed before the next S phase. The coupling between completion of DNA replication and mitosis is relatively well understood. Replicating DNA sends out a negative signal that suppresses the activation of MPF, the mitosis (or maturation) promoting factor (Dasso and Newport 1990; Smythe and Newport 1992). In contrast, little is known about other mechanisms that coordinate DNA replication with the cell cycle.Using Xenopus egg extracts, Blow and Laskey (1988) found that to get a second round of replication, nuclei either have to go through mitosis, during which the nuclear envelope breaks down and reassembles, or be permeabilized with nonionic detergents. This observation suggests the existence of an essential replication factor, called licensing factor, which is destroyed immediately after replication initiation and which cannot gain access into the nucleus unless the nuclear envelope is perturbed. In this way, cells can effectively block re-replication of their genome and then erase the block by going through mitosis. The concept of a licensing factor can easily explain the results of earlier cell fusion studies that G1 nuclei, but not G2 nuclei, can initiate DNA replication when fused with S cells (Rao and Johnson 1970). However, this hypothesis...

Mcm1 is an essential protein required for the efficient replication of minichromosomes and the transcriptional regulation of early cell cycle genes in Saccharomyces cerevisiae. In this study, we report that Mcm1 is an abundant protein that associates globally with chromatin in a punctate pattern. We show that Mcm1 is localized at replication origins and plays an important role in the initiation of DNA synthesis at a chromosomal replication origin in vivo. Using purified Mcm1 protein, we show that Mcm1 binds cooperatively to multiple sites at autonomously replicating sequences. These results suggest that, in addition to its role as a transcription factor for the expression of replication genes, Mcm1 may influence the local structure of replication origins by direct binding.