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.
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.
Masuda
Taro
T
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Mimura
Satoru
S
Takisawa
Haruhiko
H
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002457:Cell Extracts; D002843:Chromatin / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D010766:Phosphorylation; D016196:S Phase / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014981:Xenopus
C102209:CDC45 protein, S cerevisiae; D002352:Carrier Proteins; D002457:Cell Extracts; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases; D004265:DNA Helicases