The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.
The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.
Imperial Cancer Research Fund, Clare Hall Laboratories, CDC Laboratory, South Mimms, Hertfordshire EN6 3LD, UK.
J E
JE
Vialard
C S
CS
Gilbert
C M
CM
Green
N F
NF
Lowndes
D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D005656:Fungal Proteins / Q000378:metabolism*; D016195:G2 Phase / Q000235:genetics; D005720:Gamma Rays; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008938:Mitosis / Q000235:genetics; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D015739:Nocodazole; D006918:Hydroxyurea