The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding.
Eukaryotes have six minichromosome maintenance (MCM) proteins that are essential for DNA replication. The contribution of ATPase activity of MCM complexes to their function in replication is poorly understood. We have established a cell-free system competent for replication in which all MCM proteins are supplied by purified recombinant Xenopus MCM complexes. Recombinant MCM2-7 complex was able to assemble onto chromatin, load Cdc45 onto chromatin, and restore DNA replication in MCM-depleted extracts. Using mutational analysis in the Walker A motif of MCM6 and MCM7 of MCM2-7, we show that ATP binding and/or hydrolysis by MCM proteins is dispensable for chromatin loading and pre-replicative complex (pre-RC) assembly, but is required for origin unwinding during DNA replication. Moreover, this ATPase-deficient mutant complex did not support DNA replication in MCM-depleted extracts. Altogether, these results both demonstrate the ability of recombinant MCM proteins to perform all replication roles of MCM complexes, and further support the model that MCM2-7 is the replicative helicase. These data establish that mutations affecting the ATPase activity of the MCM complex uncouple its role in pre-RC assembly from DNA replication.
Ying
Carol Y
CY
Integrated Program in Cellular, Molecular, and Biophysical Studies, Hammer Health Sciences Center, Columbia University College of Physicians and Surgeons, New York, NY, 10032, USA.
Gautier
Jean
J
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002460:Cell Line; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004261:DNA Replication; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006868:Hydrolysis; D007313:Insecta; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7; D020409:Molecular Motor Proteins; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry*; D051738:Origin Recognition Complex; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D029867:Xenopus Proteins / Q000737:chemistry*; D014982:Xenopus laevis
D002352:Carrier Proteins; C502211:Cdc45 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020409:Molecular Motor Proteins; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D011506:Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; C102186:Mcm2 protein, Xenopus; C509836:Mcm7 protein, Xenopus; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7