title: Phosphorylation of Sae2 Mediates Forkhead-associated (FHA) Domain-specific Interaction and Regulates Its DNA Repair Function.
abstract: Saccharomyces cerevisiae Sae2 and its ortholog CtIP in higher eukaryotes have a conserved role in the initial processing of DNA lesions and influencing their subsequent repair pathways. Sae2 is phosphorylated by the ATR/ATM family kinases Mec1 and Tel1 in response to DNA damage. Among the Mec1/Tel1 consensus phosphorylation sites of Sae2, we found that mutations of Thr-90 and Thr-279 of Sae2 into alanine caused a persistent Rad53 activation in response to a transient DNA damage, similar to the loss of Sae2. To gain insight into the function of this phosphorylation of Sae2, we performed a quantitative proteomics analysis to identify its associated proteins. We found that phosphorylation of Thr-90 of Sae2 mediates its interaction with Rad53, Dun1, Xrs2, Dma1, and Dma2, whereas Rad53 and Dun1 additionally interact with phosphorylated Thr-279 of Sae2. Mutations of the ligand-binding residues of Forkhead-associated (FHA) domains of Rad53, Dun1, Xrs2, Dma1, and Dma2 abolished their interactions with Sae2, revealing the involvement of FHA-specific interactions. Mutations of Thr-90 and Thr-279 of Sae2 caused a synergistic defect when combined with sgs1Δ and exo1Δ and elevated gross chromosomal rearrangements. Likewise, mutations of RAD53 and DUN1 caused a synthetic growth defect with sgs1Δ and elevated gross chromosomal rearrangements. These findings suggest that threonine-specific phosphorylation of Sae2 by Mec1 and Tel1 contributes to DNA repair and genome maintenance via its interactions with Rad53 and Dun1.
authors.affiliation: From the Ludwig Institute for Cancer Research, Department of Chemistry and Biochemistry.
authors.forename: Jason
authors.initials: J
authors.lastname: Liang
authors.affiliation: From the Ludwig Institute for Cancer Research.
authors.forename: Raymond T
authors.initials: RT
authors.lastname: Suhandynata
authors.affiliation: From the Ludwig Institute for Cancer Research, Department of Chemistry and Biochemistry, Department of Cellular and Molecular Medicine, and Moores Cancer Center, University of California at San Diego, La Jolla, California 92093 huzhou@ucsd.edu.
authors.forename: Huilin
authors.initials: H
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000378:metabolism; D004260:DNA Repair; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005800:Genes, Fungal; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000633:toxicity; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D053484:RecQ Helicases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry
chemical_list: D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009153:Mutagens; C107849:SAE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D008741:Methyl Methanesulfonate; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C513623:TEL1 protein, S cerevisiae; C067022:RAD53 protein, S cerevisiae; D004720:Endonucleases; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C090472:SGS1 protein, S cerevisiae; D053484:RecQ Helicases
keywords: Checkpoint Control; DNA Damage Response; DNA Repair; Dun1; FHA Domains; Protein Phosphorylation; Protein-Protein Interaction; Rad53; Sae2; Xrs2