Affinity photo-crosslinking study of the DNA base flipping pathway by HhaI methyltransferase.
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.
Daujotyte
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D
Institute of Biotechnology, LT-2028, Vilnius, Lithuania.
Klimasauskas
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S
D000345:Affinity Labels; D001483:Base Sequence; D020134:Catalytic Domain; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases; D066298:In Vitro Techniques; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; C101233:2-thiothymine; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases; D013941:Thymine; D013936:Thymidine