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.