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CDK‐ and Cdc45‐dependent priming of the MCM complex on chromatin during S‐phase in Xenopus egg extracts: possible activation of MCM helicase by association with Cdc45
Author(s) -
Masuda Taro,
Mimura Satoru,
Takisawa Haruhiko
Publication year - 2003
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.2003.00621.x
Subject(s) - biology , origin recognition complex , minichromosome maintenance , helicase , chromatin , microbiology and biotechnology , dna replication , control of chromosome duplication , pre replication complex , replication factor c , eukaryotic dna replication , genetics , dna , gene , rna
Background: MCM and Cdc45 are required for the initiation and elongation stages of eukaryotic DNA replication. Recent studies show that a purified Mcm4/6/7 complex has DNA helicase activity. However, the biochemical function of the MCM complex and Cdc45 bound to chromatin has not been elucidated. Results: We have examined the biochemical properties of MCM proteins bound to chromatin fractions using Xenopus egg extracts. Immunoprecipitation of MCM proteins extracted under denaturing conditions reveals that all six subunits of MCM and Cdc45 form a tight complex following the initiation of DNA replication, and that both CDK activity and Cdc45 are essential for the complex formation. Chromatin immunoprecipitation of MCM proteins and Cdc45 shows that a complex containing MCM and Cdc45 has a DNA helicase activity which is dependent on CDK activity and Cdc45 in the extracts. Furthermore, both the complex and the helicase activity are resistant to treatment with phosphatase and high salt. Conclusions: Following the initiation of DNA replication, a tight MCM–Cdc45 complex is formed on chromatin and its formation is closely correlated with the DNA helicase activity of chromatin immunoprecipitates containing MCM and Cdc45. We propose that the tight MCM–Cdc45 complex functions as a replicative DNA helicase in vivo .