title: The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response to DNA damage.
abstract: Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Bethany L
authors.initials: BL
authors.lastname: Andreson
authors.forename: Amitabha
authors.initials: A
authors.lastname: Gupta
authors.forename: Bilyana P
authors.initials: BP
authors.lastname: Georgieva
authors.forename: Rodney
authors.initials: R
authors.lastname: Rothstein
mesh_terms: D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000378:metabolism; D004249:DNA Damage; D003854:Deoxyribonucleotides / Q000378:metabolism; D015536:Down-Regulation; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D002352:Carrier Proteins; D003854:Deoxyribonucleotides; C526592:Mub1 protein, S cerevisiae; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C496536:Ubr2 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease