Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

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Abstract

Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

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All data are included in the manuscript.

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Syafiq Abd Wahab

Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, United States

Competing interests
The authors declare that no competing interests exist.
Dirk Remus

Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, United States

For correspondence
remusd@mskcc.org

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-5155-181X

Funding

National Institute of General Medical Sciences (R01-GM127428)

Dirk Remus

National Institute of General Medical Sciences (R01-GM107239)

Dirk Remus

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.