title: An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.
abstract: Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication.
authors.affiliation: Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Perez-Arnaiz
authors.affiliation: Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA. Electronic address: Daniel.Kaplan@med.fsu.edu.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Kaplan
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; C088096:Dbf4 protein, S cerevisiae; C076365:MCM10 protein, S cerevisiae; C069200:MCM5 protein, S cerevisiae; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; C069199:MCM2 protein, S cerevisiae; D064110:Minichromosome Maintenance Proteins
keywords: CMG complex; DNA replication; DNA unwinding; helicase; yeast