A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication.
In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Marcin
M
Pacek
Johannes C
JC
Walter
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002875:Chromosomes / Q000502:physiology*; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000502:physiology*; D008297:Male; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism*; D018741:Replication Origin; D016160:Retinoblastoma Protein / Q000378:metabolism; D013737:Testis / Q000502:physiology; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000502:physiology*
C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016160:Retinoblastoma Protein; D029867:Xenopus Proteins; D004265:DNA Helicases; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7