title: Dpb11 protein helps control assembly of the Cdc45·Mcm2-7·GINS replication fork helicase.
abstract: Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45·Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45·Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.
authors: From the Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235 and.
authors: Nalini
authors: N
authors: Dhingra
authors: the Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida 32306-4300.
authors: Irina
authors: I
authors: Bruck
authors: the Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida 32306-4300.
authors: Skye
authors: S
authors: Smith
authors: From the Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235 and.
authors: Boting
authors: B
authors: Ning
authors: the Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida 32306-4300 Daniel.Kaplan@med.fsu.edu.
authors: Daniel L
authors: DL
authors: Kaplan
mesh_terms: D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*
chemical_list: C102209:CDC45 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C097185:DPB11 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D004265:DNA Helicases; D064110:Minichromosome Maintenance Proteins
keywords: Cell Cycle; Cyclin-dependent Kinase (CDK); DNA; DNA Helicase; DNA Replication