posted on 2000-12-19, 00:00authored byOrigène 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.