Template-directed interference footprinting of protein-phosphate contacts in DNA.
[figure: see text] We have developed a method for interference footprinting of contacted phosphates in protein-DNA complexes. Template-directed enzymatic polymerization using a synthetic triphosphate analogue (alpha Me-dTTP) generates a product having a modified Internucleotide linkage, which perturbs protein-phosphate contacts. We found that treatment of the methylphosphonodiester-substituted extension product under nonaqueous conditions (MeO-/MeOH) led to the formation of a single cleavage product at each T residue but to two cleavage products when treated under the standard aqueous piperidine cleavage protocol.
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Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
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D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D018983:DNA Footprinting / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015394:Molecular Structure; D010449:Peptide Mapping / Q000379:methods*; D010710:Phosphates / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000187:drug effects; D013329:Structure-Activity Relationship
D010710:Phosphates; D011506:Proteins; D004247:DNA