Abstract
HhaI DNA methyltransferase flips the inner cytosine in the recognition sequence 5'-GCGC-3' out of the DNA helix and into the catalytic site for methylation. To identify intermediate states on the base-flipping pathway, affinity photo-crosslinking experiments were performed with synthetic dodecamer duplexes containing modified bases 2-thiothymine (2sT) or 4-thiothymine (4sT) at the target base position. Here we show that the DNA strand containing 2sT, but not 4sT, covalently cross-links to the HhaI methyltransferase upon irradiation at 340-360 nm.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Affinity Labels
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Base Sequence
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Catalytic Domain
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Cross-Linking Reagents
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DNA / chemistry*
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DNA / metabolism*
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DNA / radiation effects
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DNA Methylation
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DNA-Cytosine Methylases*
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In Vitro Techniques
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Oligodeoxyribonucleotides / chemistry
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Oligodeoxyribonucleotides / metabolism
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Photochemistry
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Thymidine / analogs & derivatives
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Thymidine / chemistry
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Thymine / analogs & derivatives*
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Thymine / chemistry
Substances
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Affinity Labels
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Cross-Linking Reagents
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Oligodeoxyribonucleotides
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2-thiothymine
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DNA
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DNA modification methylase HhaI
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DNA-Cytosine Methylases
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Thymine
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Thymidine