title: The mini-chromosome maintenance (Mcm) complexes interact with DNA polymerase α-primase and stimulate its ability to synthesize RNA primers.
abstract: The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2~7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2~7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.
authors: Genome Dynamics Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. takai-yk@igakuken.or.jp
authors: Zhiying
authors: Z
authors: You
authors: Mariarosaria
authors: M
authors: De Falco
authors: Katsuhiko
authors: K
authors: Kamada
authors: Francesca M
authors: FM
authors: Pisani
authors: Hisao
authors: H
authors: Masai
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004265:DNA Helicases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis*
chemical_list: D004277:DNA, Single-Stranded; D046912:Multiprotein Complexes; D021122:Protein Subunits; C034477:RNA primers; D012313:RNA; D000255:Adenosine Triphosphate; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D064110:Minichromosome Maintenance Proteins
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