ATR-dependent pathways control hEXO1 stability in response to stalled forks.
Nucleases play important roles in DNA synthesis, recombination and repair. We have previously shown that human exonuclease 1 (hEXO1) is phosphorylated in response to agents stalling DNA replication and that hEXO1 consequently undergoes ubiquitination and degradation in a proteasome-dependent manner. In the present study, we have addressed the identity of the pathway transducing stalled-replication signals to hEXO1. Using chemical inhibitors, RNA interference, ATM- and ATR-deficient cell lines we have concluded that hEXO1 phosphorylation is ATR-dependent. By means of mass spectrometry, we have identified the sites of phosphorylation in hEXO1 in undamaged cells and in cells treated with hydroxyurea (HU). hEXO1 is phosphorylated at nine basal sites and three additional sites are induced by HU treatment. Analysis of single- and multiple-point mutants revealed that mutation to Ala of the three HU-induced sites of phosphorylation partially rescued HU-dependent degradation of hEXO1 and additionally stabilized the protein in non-treated cells. We have raised an antibody to pS(714), an HU-induced site of the S/T-Q type, and we provide evidence that S(714) is phosphorylated upon HU but not IR treatment. The antibody may be a useful tool to monitor signal transduction events triggered by stalled DNA replication.
Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Mahmoud
M
El-Shemerly
Daniel
D
Hess
Aswin K
AK
Pyakurel
Said
S
Moselhy
Stefano
S
Ferrari
D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004795:Enzyme Stability; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010768:Phosphoserine / Q000276:immunology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction
D045163:Antibodies, Phospho-Specific; D018797:Cell Cycle Proteins; D010768:Phosphoserine; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; D045643:DNA Repair Enzymes; D006918:Hydroxyurea