title: Molecular basis of the essential s phase function of the rad53 checkpoint kinase.
abstract: The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.
authors.affiliation: St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
authors.forename: Nicolas C
authors.initials: NC
authors.lastname: Hoch
authors.forename: Eric S-W
authors.initials: ES
authors.lastname: Chen
authors.forename: Robert
authors.initials: R
authors.lastname: Buckland
authors.forename: Shun-Chung
authors.initials: SC
authors.lastname: Wang
authors.forename: Alessandro
authors.initials: A
authors.lastname: Fazio
authors.forename: Andrew
authors.initials: A
authors.lastname: Hammet
authors.forename: Achille
authors.initials: A
authors.lastname: Pellicioli
authors.forename: Andrei
authors.initials: A
authors.lastname: Chabes
authors.forename: Ming-Daw
authors.initials: MD
authors.lastname: Tsai
authors.forename: Jörg
authors.initials: J
authors.lastname: Heierhorst
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004271:DNA, Fungal / Q000235:genetics; D004789:Enzyme Activation; D017353:Gene Deletion; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D047908:Intracellular Signaling Peptides and Proteins; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae