title: Cytoplasmic localization of Hug1p, a negative regulator of the MEC1 pathway, coincides with the compartmentalization of Rnr2p-Rnr4p.
abstract: The evolutionarily conserved MEC1 checkpoint pathway mediates cell cycle arrest and induction of genes including the RNR (Ribonucleotide reductase) genes and HUG1 (Hydroxyurea, ultraviolet, and gamma radiation) in response to DNA damage and replication arrest. Rnr complex activity is in part controlled by cytoplasmic localization of the Rnr2p-Rnr4p subunits and inactivation of negative regulators Sml1p and Dif1p upon DNA damage and hydroxyurea (HU) treatment. We previously showed that a deletion of HUG1 rescues lethality of mec1Δ and suppresses dun1Δ strains. In this study, multiple approaches demonstrate the regulatory response of Hug1p to DNA damage and HU treatment and support its role as a negative effector of the MEC1 pathway. Consistent with our hypothesis, wild-type cells are sensitive to DNA damage and HU when HUG1 is overexpressed. A Hug1 polyclonal antiserum reveals that HUG1 encodes a protein in budding yeast and its MEC1-dependent expression is delayed compared to the rapid induction of Rnr3p in response to HU treatment. Cell biology and subcellular fractionation experiments show localization of Hug1p-GFP to the cytoplasm upon HU treatment. The cytoplasmic localization of Hug1p-GFP is dependent on MEC1 pathway genes and coincides with the cytoplasmic localization of Rnr2p-Rnr4p. Taken together, the genetic interactions, gene expression, and localization studies support a novel role for Hug1p as a negative regulator of the MEC1 checkpoint response through its compartmentalization with Rnr2p-Rnr4p.
authors.affiliation: Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803, USA.
authors.forename: William B
authors.initials: WB
authors.lastname: Ainsworth
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Bridget Todd
authors.initials: BT
authors.lastname: Hughes
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Wei Chun
authors.initials: WC
authors.lastname: Au
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Sally
authors.initials: S
authors.lastname: Sakelaris
authors.affiliation: Biology Department, The College of William & Mary, Williamsburg, VA 23185, USA.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kerscher
authors.affiliation: Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Benton
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Munira A
authors.initials: MA
authors.lastname: Basrai
mesh_terms: D003593:Cytoplasm / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C403293:HUG1 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C570014:RNR2 protein, S cerevisiae; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; C523202:Rnr4 protein, S cerevisiae; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases
keywords: BLM; DEX; DNA damage; GAL; GFP; HU; HU arrest; HUG1; MEC1; MMS; PBS; RNR; Rnr; Saccharomyces cerevisiae; Subcellular localization; bleomycin; dNTP; deoxyribonucleotide; dextrose; galactose; green fluorescent protein; hydroxyurea; methyl methanesulfonate; phosphate buffered saline; ribonucleotide reductase