High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site.
The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.
Mernagh
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DR
Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Hants, UK.
Kneale
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GG
D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000235:genetics / Q000378:metabolism*; D017665:Hydroxyl Radical; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D017665:Hydroxyl Radical; D004247:DNA; C048242:endodeoxyribonuclease EcoR124I; D003850:Deoxyribonuclease I; D015253:Deoxyribonucleases, Type I Site-Specific