The methyl methanesulfonate induced S-phase delay in XRCC1-deficient cells requires ATM and ATR.
X-ray repair cross-complementing 1 (XRCC1) is required for DNA single-strand break and base excision repair (BER) in human cells. XRCC1-deficient human cells show hypersensitivity to cell killing, increased genetic instability and a significant delay in S-phase progression after exposure to the alkylating agent methyl methanesulfonate (MMS). Using RNAi modulation of XRCC1 levels, we show here that this S-phase delay is associated with significantly increased levels of recombinational repair as visualized by Rad51 focus formation. Using ATM- and ATR-defective cells and an ATM specific kinase inhibitor we demonstrate for the first time that the MMS-induced S-phase checkpoint requires both ATM and ATR. This unique dependency is associated with phosphorylation of ATM/ATR downstream targets or effectors such as SMC1 and Chk1. These results support the hypothesis that after MMS-treatment, the presence of unresolved BER intermediates gives rise to lesions that activate both ATM and ATR and that during the consequent S-phase delay, such intermediates may be repaired by a recombinational pathway which involves the Rad51 protein.
Brem
Reto
R
International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon, France.
Fernet
Marie
M
Chapot
Brigitte
B
Hall
Janet
J
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D034622:RNA Interference; D016196:S Phase / Q000187:drug effects*; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology*; D000076105:X-ray Repair Cross Complementing Protein 1
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D008741:Methyl Methanesulfonate; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases