Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast.
Fission yeast Mrc1 (mediator of replication checkpoint 1) is an adaptor checkpoint protein required for Rad3-dependent activation of the checkpoint kinase Cds1 in response to arrest of replication forks. Here we report studies on the regulation of Mrc1 by phosphorylation. Replication arrest induced by hydroxyurea (HU) induces Mrc1 phosphorylation that is detected by a change in Mrc1 electrophoretic mobility. Phosphorylation is maintained in cds1Delta, rad3Delta, and tel1Delta single mutants but eliminated in a rad3Delta tel1Delta double mutant. Mrc1 has two clusters of S/TQ motifs that are potential Rad3/Tel1 phosphorylation sites. Mutation of six S/TQ motifs in these two clusters strongly impairs Mrc1 phosphorylation. Two motifs located at S604 and T645 are vital for HU resistance. The T645A mutation strongly impairs a Cds1-Mrc1 yeast two-hybrid interaction that is dependent on a functional forkhead-associated (FHA) domain in Cds1, indicating that phosphorylation of T645 mediates Mrc1's association with Cds1. Consistent with this model, the T645 region of Mrc1 effectively substitutes for the T11 region of Cds1 that is thought to be phosphorylated by Rad3 and to mediate FHA-dependent oligomerization of Cds1. The S/TQ cluster that includes S604 is needed for Mrc1's increased association with chromatin in replication-arrested cells. These data indicate that Rad3 and Tel1 regulate Mrc1 through differential phosphorylation to control Cds1.
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Hui
H
Zhao
Katsunori
K
Tanaka
Eishi
E
Nogochi
Chiaki
C
Nogochi
Paul
P
Russell
D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004271:DNA, Fungal / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D004271:DNA, Fungal; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C441579:MRC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; C513623:TEL1 protein, S cerevisiae; C456840:rad3 protein, S pombe; C513624:tel1 protein, S pombe; D006918:Hydroxyurea