Mec1 is one of multiple kinases that prime the Mcm2-7 helicase for phosphorylation by Cdc7.
Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms that modulate origin firing and replication fork assembly during cell cycle progression.
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
John C W
JC
Randell
Andy
A
Fan
Clara
C
Chan
Laura I
LI
Francis
Ryan C
RC
Heller
Kyriaki
K
Galani
Stephen P
SP
Bell
D000595:Amino Acid Sequence; D000596:Amino Acids; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D064168:Minichromosome Maintenance Complex Component 7; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C489404:CDC7 protein, S cerevisiae; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C069199:MCM2 protein, S cerevisiae; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7