The Molecular Mechanism Of DDK Kinase In Control Of DNA Replication Initiation And Timing In Saccharomyces Cerevisiae

DNA replication ensures the accurate duplication and transmission of genetic information in all types of cells.Eukaryotic DNA replication initiates from hundreds and thousands of origins which is highly coordinated in a spatial-temporal manner during the S-phase of cell cycle.In Saccharomyces cerevisiae,DDK(Dbf4-dependent kinase)is involved in Mcm2-7 helicase activation during DNA replication initiation.Meanwhile,DDK is also an important limiting factor during replication timing control,its regulatory subunit Dbf4 is preferentially targeted to early origins and overexpression of DDK causes many late origins firing in early S phase.However,how Dbf4 is preferentially recruited to early origins remains obscure.This paper is mainly aimed to explore the regulation mechanism of DDK in replication initiation and timing control.First,through yeast two hybrid screening,we identified two Dbf4 interactors,transcription factors Fkhl and Fkh2.The physical associations were further confirmed by coimmunoprecipitation.Moreover,when Fkhl and Fkh2 were deleted,enrichment of Dbf4 at early origins was significantly decreased and the firing of early origins was also delayed.The C-terminus of Dbf4 mediates the interaction between Dbf4 and Fkhl/Fkh2,and the firing of early origins was significantly delayed in the absence of Dbf4 C-terminus.The replication timing program of dbf4AC can be rescued to be neally wild-type though direct fusing Dbf4 with the forkhead domain of Fkhl.All these results allow us to conclude that Fkhl and Fkh2 regulate replication timing by direct recruiting Dbf4 to early origins.Second,in the yeast two hybrid screen,we also noticed positive interactions of Sld3 with Mcm2 and Mcm6.The SId3-binding domain(SBD)is mapped to the N termini of Mcm2 and Mcm6.Interestingly,both of them are essential for cell viability and enriched with DDK phosphorylation sites.Glutamic acid substitution of four conserved positively charged amino acids of Sld3 interrupts its interaction with Mcm2/Mcm6 and causes cell death.By using a temperature-inducible degron(td),we showed that deletion of Mcm6 SBD(mcm6?N122)abolishes not only Sld3 enrichment at early origins in G1 phase,but also subsequent recruitment of GINS and RPA during S phase.Therefore,these results illustrate the molecular details of the DDK-dependent Sld3-MCM association,which plays a crucial role in MCM helicase activation and origin unwinding.Dysregulation of DNA replication timing correlates with genome instability and tumorigenesis.The molecular mechanism of DDK in replication initiation and timing control elucidated here will help us to understand how this fundamental process in cell fate decision is properly controlled in eukaryotes.