Analysis of Cdc6 function in the assembly of mammalian prereplication complexes.
Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex. Although considerable insight has been gained from in vitro studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking. We have made use of adenoviral vectors to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function. We demonstrate that Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low. Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells. Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6. Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication. Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication. Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6.
Cook
Jeanette Gowen
JG
Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Box 3054, Durham, NC 27710, USA.
Park
Chi-Hyun
CH
Burke
Thomas W
TW
Leone
Gustavo
G
DeGregori
James
J
Engel
Amber
A
Nevins
Joseph R
JR
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000906:Antibodies; D001973:Bromodeoxyuridine; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D002843:Chromatin / Q000235:genetics / Q000502:physiology; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication; D008322:Mammals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D000906:Antibodies; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D019927:Cyclin E; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D001973:Bromodeoxyuridine; D013936:Thymidine