Abstract
S(N)1-type alkylating agents that produce cytotoxic O(6)-methyl-G (O(6)-meG) DNA adducts induce cell cycle arrest and apoptosis in a manner requiring the DNA mismatch repair (MMR) proteins MutSalpha and MutLalpha. Here, we show that checkpoint signaling in response to DNA methylation occurs during S phase and requires DNA replication that gives rise to O(6)-meG/T mispairs. DNA binding studies reveal that MutSalpha specifically recognizes O(6)-meG/T mispairs, but not O(6)-meG/C. In an in vitro assay, ATR-ATRIP, but not RPA, is preferentially recruited to O(6)-meG/T mismatches in a MutSalpha- and MutLalpha-dependent manner. Furthermore, ATR kinase is activated to phosphorylate Chk1 in the presence of O(6)-meG/T mispairs and MMR proteins. These results suggest that MMR proteins can act as direct sensors of methylation damage and help recruit ATR-ATRIP to sites of cytotoxic O(6)-meG adducts to initiate ATR checkpoint signaling.
Publication types
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Research Support, N.I.H., Intramural
MeSH terms
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Adaptor Proteins, Signal Transducing
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Animals
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Ataxia Telangiectasia Mutated Proteins
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Base Pair Mismatch
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Base Sequence
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Carrier Proteins / metabolism
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Cell Cycle Proteins / metabolism*
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Cell Line
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DNA / genetics
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DNA / metabolism
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DNA Adducts / metabolism*
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DNA Adducts / toxicity
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DNA Damage
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DNA Repair / physiology*
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DNA-Binding Proteins
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Enzyme Activation / drug effects
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Exodeoxyribonucleases / metabolism
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Guanine / analogs & derivatives*
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Guanine / metabolism
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HeLa Cells
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Humans
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Mice
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MutS DNA Mismatch-Binding Protein / metabolism*
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Nuclear Proteins / metabolism
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Phosphoproteins / metabolism
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Protein Serine-Threonine Kinases / metabolism*
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S Phase
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Signal Transduction
Substances
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ATRIP protein, human
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Adaptor Proteins, Signal Transducing
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Carrier Proteins
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Cell Cycle Proteins
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DNA Adducts
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DNA-Binding Proteins
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Nuclear Proteins
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Phosphoproteins
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Guanine
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DNA
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O-(6)-methylguanine
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ATR protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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Exodeoxyribonucleases
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three prime repair exonuclease 1
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MutS DNA Mismatch-Binding Protein