title: Structural insights into the Cdt1-mediated MCM2-7 chromatin loading.
abstract: Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.
authors.affiliation: Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: Changdong
authors.initials: C
authors.lastname: Liu
authors.forename: Rentian
authors.initials: R
authors.lastname: Wu
authors.forename: Bo
authors.initials: B
authors.lastname: Zhou
authors.forename: Jiafeng
authors.initials: J
authors.lastname: Wang
authors.forename: Zhun
authors.initials: Z
authors.lastname: Wei
authors.forename: Bik K
authors.initials: BK
authors.lastname: Tye
authors.forename: Chun
authors.initials: C
authors.lastname: Liang
authors.forename: Guang
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D051379:Mice; D064151:Minichromosome Maintenance Complex Component 6; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; C103258:MCM6 protein, human; D064151:Minichromosome Maintenance Complex Component 6