Localization of MCM2-7, Cdc45, and GINS to the Site of DNA Unwinding during Eukaryotic DNA Replication

• Published in: Molecular cell 2006-02, Vol.21 (4), p.581-587
• Main Authors: Pacek, Marcin, Tutter, Antonin V., Kubota, Yumiko, Takisawa, Haruhiko, Walter, Johannes C.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Animals; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; DNA; DNA - chemistry; DNA - metabolism; DNA Replication; DNA-Directed DNA Polymerase - metabolism; Macromolecular Substances; Minichromosome Maintenance Complex Component 2; Minichromosome Maintenance Complex Component 3; Minichromosome Maintenance Complex Component 7; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nucleic Acid Conformation; Xenopus; Xenopus Proteins - genetics; Xenopus Proteins - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2006.01.030

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 α, DNA pol δ, DNA pol ɛ, 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.