Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase

Julia F. Greiwe; Thomas C.R. Miller; Julia Locke; Fabrizio Martino; Steven Howell; Anne Schreiber; Andrea Nans; John F.X. Diffley; Alessandro Costa

doi:10.1038/s41594-021-00698-z

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase

Julia F. Greiwe, Thomas C.R. Miller, Julia Locke, Fabrizio Martino, Steven Howell, Anne Schreiber, Andrea Nans, John F.X. Diffley, Alessandro Costa^*

^*Corresponding author af dette arbejde

Molecular Aging Program

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

32 Citationer (Scopus)

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Abstract

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

Originalsprog	Engelsk
Tidsskrift	Nature Structural and Molecular Biology
Vol/bind	29
Sider (fra-til)	10-20
Antal sider	11
ISSN	1545-9993
DOI	https://doi.org/10.1038/s41594-021-00698-z
Status	Udgivet - 2022

Bibliografisk note

Funding Information:
Cherepanov for sharing unpublished data and for the gift of the MCM peptide; and P. Walker, A. Purkiss, A. Alidoust, N. Patel and D. Patel (Structural Biology STP) for help with EM access, computing and protein expression. Work at the Crick is funded jointly by the Wellcome Trust, MRC and CRUK at the Francis Crick Institute (nos. FC001065 to A.C., FC001066 to J.F.X.D. and FC001598 to A.S.). Work in A.C.’s laboratory is funded by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 820102). Work in J.F.X.D.’s laboratory is also funded by a Wellcome Trust Senior Investigator award (no. 106252/Z/14/Z) and a European Research Council Advanced Grant (no. 669424-CHROMOREP).

Publisher Copyright:
© 2021, The Author(s).

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Citationsformater

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title = "Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase",

abstract = "Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.",

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T1 - Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase

AU - Greiwe, Julia F.

AU - Miller, Thomas C.R.

AU - Locke, Julia

AU - Martino, Fabrizio

AU - Howell, Steven

AU - Schreiber, Anne

AU - Nans, Andrea

AU - Diffley, John F.X.

AU - Costa, Alessandro

PY - 2022

Y1 - 2022

N2 - Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

AB - Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

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DO - 10.1038/s41594-021-00698-z

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SN - 1545-9993

VL - 29

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JO - Nature Structural and Molecular Biology

JF - Nature Structural and Molecular Biology

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