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Template-Directed Interference Footprinting of Protein−Phosphate Contacts in DNA

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journal contribution
posted on 2000-12-19, 00:00 authored by Origène Nyanguile, Gregory L. Verdine
We have developed a method for interference footprinting of contacted phosphates in protein−DNA complexes. Template-directed enzymatic polymerization using a synthetic triphosphate analogue (αMe-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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