The Molecular Mechanisms Of The Activation Of DNA Replicative Helicase Mcm2-7 By Mcm10 In Saccharomyces Cerevisiae

DNA replication is one of the fundamental and essential processes in all living species on earth.The accurate copying of genetic information is essential for cell growth and organism development.Errors and disorders during DNA replication can manifest as genomic instability and result in genetic diseases,cancers,developmental abnormalities,reproductive deficiencies and even death.Understanding the molecular mechanisms of DNA replication is therefore critical for basic life sciences as well as diagnosis and therapy for a variety of human genomic instability related diseases.The assembly of the DNA replication machinery and initiation of synthesis are controlled in a tightly orchestrated manner according to different stages of the cell cycle.Mcm2-7 helicase is loaded onto double stranded DNA as an inactive double hexamer(DH)during G1 phase.When cells enter S phase,the levels of essential kinases S-CDK and DDK increase and promote the recruitment of Cdc45 and GINS,two co-activators of Mcm2-7,onto MCM DH,thereby forming two acitive Cdc45-Mcm2-7-GINS complexes.However,the underlying mechanisms of Mcm2-7 remodeling to trigger helicase activation in the S phase remains elusive.Mcm 10,one of the essential firing factors,has been inferred to be important in Mcm2-7 helicase activation.However,the molecular underpinnings of Mcm10's function have yet to be defined.To uncover the essential role of Mcm10 in replication initiation,we have investigated the interaction between Mcm10 and Mcm2-7 systemically in Saccharomyces cerevisiae.Mcm10 preferentially associates with Mcm2-7 DH in the context of chromatin,despite its direct binding to Mcm2-7 subuinits in vitro.We show that Mcm10 binds robustly to the Mcm2/4/6 N-termini,indicating it binds to near the interface of MCM DH.Moreover,the Mcm10-DH interaction requires the Mcm10 C-terminus and is facilitated by Mcm10 phosphorylation at a conserved CDK site,S66.mcmlOAC-S66 alleles which specifically abolish MCM interaction are lethal,but suppressed by Mcm10-MCM fusions.Taken together,the Mcm10-DH interaction is essential for cell growth and DNA replication initiation.Functional interaction and in vivo tethering analysis suggest an unanticipated crosstalk between Mcm10 and Dbf4-dependent kinase(DDK),which has been identified as another essential requirement for helicase activation.The two MCM rings are dimerized through an interface composed of the N termini of Mcm2-7 subunits,which bears multiple critical target sites for DDK.We show that Mcm10 binds both to the Mcm2/4/6 N-termini and the DDK subunits,suggesting Mcm10 and DDK facilitate Mcm2-7 activation synergistically.However,we find that the phosphorylation of Mcm4 by DDK and subsequent recruitment of Cdc45 are not dependent on Mcm10-MCM interaction,implying the role of this interaction lies in MCM DH separation.To monitor the DH sepatation in vivo,we establish a new approach to quantify the relative amounts of DH in vivo,providing the first direct evidence of the crucial role of Mcm10 in remodeling DHs into single hexamers(SHs)when cells enter S phase.We also show a significant delay in DH splitting during S phase in the Mcm10-MCM DH interaction-deficient mutants,indicating a critical role for Mcm10 in DH splitting and activation is due to the interaction between Mcm10 and MCM DH.The structure of DNA helicase has been well studied through reconsititution by purified proteins in vitro.Through the in vivo DH splitting assay,we can detect the dynamics of DH formation and separation.Importantly,we have studied the Mcm10-Mcm2-7 interaction systermatically as well as its regulation,providing novel insight into the molecular mechanisms of Mcm10 in remodeling the Mcm2-7 DHs into SHs,which are dependent on Mcm10-Mcm2-7 interaction.Our work provide a clearer model for bidirectional DNA replication.