The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1.
Cell cycle checkpoints are evolutionarily conserved surveillance systems that protect genomic stability and prevent oncogenesis in mammals. One important target of checkpoint control is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP and DNA synthesis. In both yeast and humans, RNR is transcriptionally induced after DNA damage via Mec1/Rad53 (yeast) and ATM/CHK2 (human) checkpoint pathways. In addition, yeast checkpoint proteins Mec1 and Rad53 also regulate the RNR inhibitor Sml1. After DNA damage or at S phase, Mec1 and Rad53 control the phosphorylation and concomitant degradation of Sml1 protein. This new layer of control contributes to the increased dNTP production likely necessary for DNA repair and replication; however, the molecular mechanism is unclear. Here we show that Dun1, a downstream kinase of Mec1/Rad53, genetically and physically interacts with Sml1 in vivo. The absence of Dun1 activity leads to the accumulation of Sml1 protein at S phase and after DNA damage. As a result, dun1Delta strains need more time to finish DNA replication, are defective in mitochondrial DNA propagation, and are sensitive to DNA-damaging agents. Moreover, phospho-Sml1 is absent or dramatically reduced in dun1Delta cells. Finally, Dun1 can phosphorylate Sml1 in vitro. These results suggest that Dun1 kinase function is the last step required in the Mec1/Rad53 cascade to remove Sml1 during S phase and after DNA damage.
Zhao
Xiaolan
X
Department of Genetics and Development, Columbia University, College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032-2704, USA.
Rothstein
Rodney
R
D000483:Alleles; D018797:Cell Cycle Proteins; D002455:Cell Division; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000235:genetics; D004791:Enzyme Inhibitors; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005804:Genes, Lethal / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D010641:Phenotype; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D000071164:Trefoil Factor-2; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C000604007:TFF2 protein, human; D000071164:Trefoil Factor-2; D012264:Ribonucleotide Reductases; D011494:Protein Kinases; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae