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

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

doi:10.1038/s41594-021-00698-z

Julia F Greiwe, Julia Locke + Show 7 more

Open Access

https://doi.org/10.1038/s41594-021-00698-z

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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.

Highlights

DNA replication is tightly regulated to ensure that one accurate copy of each chromosome is inherited by two daughter cells[1]
These include DDK and cyclin-dependent kinase (CDK) required for replication fork establishment, and S phase checkpoint kinases that block late origin firing if DNA damage is detected[6,7,8,9,10,11]
Our finding suggests that adenosine diphosphate (ADP) might be released and a new ATP molecule bound to the Mcm[] active site—for example, after loading of a first MCM ring and before double hexamer (DH) formation

Summary

Results

This structural flexibility allows DDK to visit an extended configuration, where Dbf[4] BRCT touches Mcm[2] on one ring (which we refer to as in cis) while Cdc[7] phosphorylates the juxtaposed Mcm[4] in trans, as observed in a composite map that combines LAFTER-filtered BRCT Dbf[4] density with the DH structure bound by the DDK core particle (Fig. 4a) or depicted in the interactome map (Fig. 4b) Such architecture immediately suggests a mechanism for how DDK derives specificity for the DNA-loaded DH, by recognizing its 3D shape. Inhibition is probably due to the cumulative effect of the 19 Rad[53] phosphosites spanning the length of the Dbf[4] molecule

Discussion

Findings

Methods