A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase

Nucleic Acids Research
M Carmen HerreraChristian Speck

Abstract

During G1-phase of the cell-cycle the replicative MCM2-7 helicase becomes loaded onto DNA into pre-replicative complexes (pre-RCs), resulting in MCM2-7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number of helicase-activation factors the assembly of a Cdc45-MCM2-7-GINS (CMG) complex. However, in the absence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2-7 double-hexamer represents a very large interaction surface. Currently it is unclear what mechanisms restricts complex assembly and how DDK can overcome this inhibition to promote CMG-assembly. We developed an advanced reconstituted-system to study helicase activation in-solution and discovered that individual factors like Sld3 and Sld2 can bind directly to the pre-RC, while Cdc45 cannot. When Sld3 and Sld2 were incubated together with the pre-RC, we observed that competitive interactions restrict complex assembly. DDK stabilizes the Sld3/Sld2-pre-RC complex, but the complex is only short-lived, indicating an anti-cooperative mechanism. Yet, a Sld3/Cdc45-pre-RC can form in the presence of DDK and the addition of Sld2 enhances complex stability. Our results i...Continue Reading

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Citations

Dec 29, 2015·Nucleus·Irina BruckDaniel L Kaplan
Dec 28, 2016·Genes· Larasati, Bernard P Duncker
Oct 29, 2017·Proceedings of the National Academy of Sciences of the United States of America·Yasunori NoguchiHuilin Li
Jul 19, 2017·Genes & Development·Alberto RieraChristian Speck

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Methods Mentioned

BETA
gel-filtration
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