title: Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.
abstract: The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory, South Mimms Herts, UK.
authors.forename: Tom D
authors.initials: TD
authors.lastname: Deegan
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory, South Mimms Herts, UK.
authors.forename: Joseph Tp
authors.initials: JT
authors.lastname: Yeeles
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory, South Mimms Herts, UK john.diffley@crick.ac.uk.
authors.forename: John Fx
authors.initials: JF
authors.lastname: Diffley
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D064148:Minichromosome Maintenance Complex Component 4 / Q000378:metabolism*; D064151:Minichromosome Maintenance Complex Component 6 / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: C102209:CDC45 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins; C426020:Sld3 protein, S cerevisiae; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C092802:MCM4 protein, S cerevisiae; C491207:MCM6 protein, S cerevisiae; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6
keywords: DDK; DNA replication initiation; Sld3