title: Cdt1 forms a complex with the minichromosome maintenance protein (MCM) and activates its helicase activity.
abstract: Mcm4/6/7 forms a complex possessing DNA helicase activity, suggesting that Mcm may be a central component for the replicative helicase. Although Cdt1 is known to be essential for loading of Mcm onto the chromatin, its precise role in pre-RC formation and replication initiation is unknown. Using purified proteins, we show that Cdt1 forms a complex with Mcm4/6/7, Mcm2/3/4/5/6/7, and Mcm2/4/6/7 in glycerol gradient fractionation through interaction with Mcm2 and Mcm4/6. In the glycerol gradient fractionation, Mcm4/6/7-Cdt1 forms a complex (speculated to be a (Mcm4/6/7)2-Cdt13 assembly) in the presence of ATP, which is significantly larger than the Mcm4/6/7-Cdt1 complex generated in its absence. Furthermore, DNA binding and helicase activities of Mcm4/6/7 are significantly stimulated by Cdt1 protein in vitro. We generated a Cdt1 mutant, which fails to stimulate DNA binding and helicase activities of Mcm4/6/7. This mutant Cdt1 showed reduced interaction with Mcm and is deficient in the formation of a high molecular weight complex with Mcm. Thus, a productive interaction between Cdt1 and MCM appears to be essential for efficient loading of MCM onto template DNA, as well as for the efficient unwinding reaction.
authors.affiliation: Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, 18-22 Honkomagome 3-chome, Bunkyo-ku, Tokyo 113-8613, Japan.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: You
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C498645:Ris2 protein, mouse; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; C516793:Mcm4 protein, mouse; C491205:Mcm6 protein, mouse; C491086:Mcm7 protein, mouse; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7