Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication.
Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Marcin
M
Pacek
Antonin V
AV
Tutter
Yumiko
Y
Kubota
Haruhiko
H
Takisawa
Johannes C
JC
Walter
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D046911:Macromolecular Substances; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; C502211:Cdc45 protein, Xenopus; D018797:Cell Cycle Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D029867:Xenopus Proteins; C489425:mcm5 protein, Xenopus; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; C507762:MCM3 protein, Xenopus; C102186:Mcm2 protein, Xenopus; C509836:Mcm7 protein, Xenopus; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064168:Minichromosome Maintenance Complex Component 7