title: CDK prevents Mcm2-7 helicase loading by inhibiting Cdt1 interaction with Orc6.
abstract: In Saccharomyces cerevisiae cells, B-type cyclin-dependent kinases (CDKs) target two origin recognition complex (ORC) subunits, Orc2 and Orc6, to inhibit helicase loading. We show that helicase loading by ORC is inhibited by two distinct CDK-dependent mechanisms. Independent of phosphorylation, binding of CDK to an "RXL" cyclin-binding motif in Orc6 sterically reduces the initial recruitment of the Cdt1/Mcm2-7 complex to ORC. CDK phosphorylation of Orc2 and Orc6 inhibits the same step in helicase loading. This phosphorylation of Orc6 is stimulated by the RXL motif and mediates the bulk of the phosphorylation-dependent inhibition of helicase loading. Direct binding experiments show that CDK phosphorylation specifically blocks one of the two Cdt1-binding sites on Orc6. Consistent with the inactivation of one Cdt1-binding site preventing helicase loading, CDK phosphorylation of ORC causes a twofold reduction of initial Cdt1/Mcm2-7 recruitment but results in nearly complete inhibition of Mcm2-7 loading. Intriguingly, in addition to being a target of both CDK inhibitory mechanisms, the Orc6 RXL/cyclin-binding motif plays a positive role in the initial recruitment of Cdt1/Mcm2-7 to the origin, suggesting that this motif is critical for the switch between active and inhibited ORC function at the G1-to-S-phase transition.
authors.affiliation: Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Shuyan
authors.initials: S
authors.lastname: Chen
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bell
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000037:antagonists & inhibitors / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D064168:Minichromosome Maintenance Complex Component 7; D008954:Models, Biological; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D030781:Organisms, Genetically Modified; D051738:Origin Recognition Complex / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C499592:TAH11 protein, S cerevisiae; D018844:Cyclin-Dependent Kinases; C069199:MCM2 protein, S cerevisiae; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7