title: Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase.
abstract: Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase.
authors: Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306.
authors: Irina
authors: I
authors: Bruck
authors: Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306 Daniel.Kaplan@med.fsu.edu.
authors: Daniel L
authors: DL
authors: Kaplan
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2 / Q000235:genetics / Q000378:metabolism*; D064110:Minichromosome Maintenance Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
chemical_list: C102209:CDC45 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C426020:Sld3 protein, S cerevisiae; C547406:TICRR protein, human; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D064111:Minichromosome Maintenance Complex Component 2; D064110:Minichromosome Maintenance Proteins
keywords: DNA replication; helicase; initiation; kinase; phosphorylation