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Analysis of Cdc6 function in the assembly of mammalian prereplication complexes.

Publication ,  Journal Article
Cook, JG; Park, C-H; Burke, TW; Leone, G; DeGregori, J; Engel, A; Nevins, JR
Published in: Proceedings of the National Academy of Sciences of the United States of America
February 2002

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.

Duke Scholars

Published In

Proceedings of the National Academy of Sciences of the United States of America

DOI

EISSN

1091-6490

ISSN

0027-8424

Publication Date

February 2002

Volume

99

Issue

3

Start / End Page

1347 / 1352

Related Subject Headings

  • Thymidine
  • Recombinant Proteins
  • Protein Serine-Threonine Kinases
  • Phosphorylation
  • Nuclear Proteins
  • Mammals
  • DNA Replication
  • Cyclin-Dependent Kinases
  • Cyclin-Dependent Kinase 2
  • Cyclin E
 

Citation

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ICMJE
MLA
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Cook, J. G., Park, C.-H., Burke, T. W., Leone, G., DeGregori, J., Engel, A., & Nevins, J. R. (2002). Analysis of Cdc6 function in the assembly of mammalian prereplication complexes. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1347–1352. https://doi.org/10.1073/pnas.032677499
Cook, Jeanette Gowen, Chi-Hyun Park, Thomas W. Burke, Gustavo Leone, James DeGregori, Amber Engel, and Joseph R. Nevins. “Analysis of Cdc6 function in the assembly of mammalian prereplication complexes.Proceedings of the National Academy of Sciences of the United States of America 99, no. 3 (February 2002): 1347–52. https://doi.org/10.1073/pnas.032677499.
Cook JG, Park C-H, Burke TW, Leone G, DeGregori J, Engel A, et al. Analysis of Cdc6 function in the assembly of mammalian prereplication complexes. Proceedings of the National Academy of Sciences of the United States of America. 2002 Feb;99(3):1347–52.
Cook, Jeanette Gowen, et al. “Analysis of Cdc6 function in the assembly of mammalian prereplication complexes.Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 3, Feb. 2002, pp. 1347–52. Epmc, doi:10.1073/pnas.032677499.
Cook JG, Park C-H, Burke TW, Leone G, DeGregori J, Engel A, Nevins JR. Analysis of Cdc6 function in the assembly of mammalian prereplication complexes. Proceedings of the National Academy of Sciences of the United States of America. 2002 Feb;99(3):1347–1352.
Journal cover image

Published In

Proceedings of the National Academy of Sciences of the United States of America

DOI

EISSN

1091-6490

ISSN

0027-8424

Publication Date

February 2002

Volume

99

Issue

3

Start / End Page

1347 / 1352

Related Subject Headings

  • Thymidine
  • Recombinant Proteins
  • Protein Serine-Threonine Kinases
  • Phosphorylation
  • Nuclear Proteins
  • Mammals
  • DNA Replication
  • Cyclin-Dependent Kinases
  • Cyclin-Dependent Kinase 2
  • Cyclin E