DNA unwinding is an Mcm complex-dependent and ATP hydrolysis-dependent process.
Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activities in vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) from Xenopus laevis eggs and six purified polyclonal antibodies generated against each of the Xenopus Mcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2-7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21(cip1) specifically blocks origin firing but does not prevent helicase action. When p21(cip1) is added, followed by the non-hydrolyzable analog ATPgammaS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.
Integrated Program in Cellular, Molecular, and Biophysical Studies, Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
David
D
Shechter
Carol Y
CY
Ying
Jean
J
Gautier
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000502:physiology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000502:physiology; D004265:DNA Helicases / Q000502:physiology; D004261:DNA Replication; D006868:Hydrolysis; D064111:Minichromosome Maintenance Complex Component 2; D011817:Rabbits; D013698:Templates, Genetic; D014981:Xenopus; D029867:Xenopus Proteins / Q000502:physiology*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D029867:Xenopus Proteins; C489425:mcm5 protein, Xenopus; C491206:mcm6a protein, Xenopus; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C102186:Mcm2 protein, Xenopus; D064111:Minichromosome Maintenance Complex Component 2