Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts.
BACKGROUND
In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood.


RESULTS
Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase alpha and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase epsilon onto chromatin required Cdc45 but did not require DNA polymerase alpha. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase alpha on chromatin.


CONCLUSIONS
These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA.
Mimura
S
S
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Masuda
T
T
Matsui
T
T
Takisawa
H
H
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D046911:Macromolecular Substances; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D018741:Replication Origin / Q000502:physiology*; D051716:Replication Protein A; D016196:S Phase / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014981:Xenopus / Q000378:metabolism*
C102209:CDC45 protein, S cerevisiae; D002352:Carrier Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D004256:DNA Polymerase I; D004257:DNA Polymerase II