A Lesion in the DNA Replication Initiation Factor Mcm10 Induces Pausing of Elongation Forks through Chromosomal Replication Origins in Saccharomyces cerevisiae

Abstract

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 ...

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

“…Mcm10 is essential for DNA replication in yeast (Solomon et al, 1992;Merchant et al, 1997), and it is likely that this extends to higher eukaryotes as well, although this has not been experimentally confirmed. To define the role of human Mcm10 in DNA replication, we used a siRNA knockdown strategy to deplete Mcm10 in HeLa cells.…”

“…The association of Cdc45 and GINS with DNA is interdependent (Kubota et al, 2003;Takayama et al, 2003), and it requires yet another factor, Mcm10 (Wohlschlegel et al, 2002;Gregan et al, 2003;Sawyer et al, 2004). Despite its name, Mcm10 is not a member of the MCM protein family, although it was isolated in the same genetic screen in budding yeast that identified the MCM2-7 genes (Solomon et al, 1992;Merchant et al, 1997).…”

“…Mcm10 is a constitutively nuclear DNA binding protein (Merchant et al, 1997;Burich and Lei, 2003) that is essential in yeast (Burich and Lei, 2003). Besides its role in DNA replication, Mcm10 has also been implicated in transcriptional silencing (Liachko and Tye, 2005).…”

“…The association of Cdc45 and GINS with DNA is interdependent (Kubota et al, 2003;Takayama et al, 2003), and it requires yet another factor, Mcm10 (Wohlschlegel et al, 2002;Gregan et al, 2003;Sawyer et al, 2004). Despite its name, Mcm10 is not a member of the MCM protein family, although it was isolated in the same genetic screen in budding yeast that identified the MCM2-7 genes (Solomon et al, 1992;Merchant et al, 1997).Mcm10 is a constitutively nuclear DNA binding protein (Merchant et al, 1997;Burich and Lei, 2003) that is essential in yeast (Burich and Lei, 2003). Besides its role in DNA replication, Mcm10 has also been implicated in transcriptional silencing (Liachko and Tye, 2005).…”

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…(66,67) In S. cerevisiae, Mcm10 associates with Mcm2-Mcm7 proteins and with the nuclear chromatin. (68) A mutant MCM10 allele was defective in initiation of replication at the ARS1 origin and also induced the pausing of replication forks coming from neighboring origins. (69) Both defects were suppressed by certain mutations in MCM5 (mcm5-461) or MCM7 (mcmc7-1).…”