Sequence-specific cleavage of DNA by N-bromoacetyldistamycin. Product and kinetic analyses

Abstract

N-Bromoacetyldistamycin (BO) is a designed molecule with two structural domains with distinct functions: sequence specific binding to double helical DNA and cleavage of the DNA backbone. An electrophilic bromoacetyl group is appended to the amino end of the tripeptide from the natural product distamycin A. Footprinting studies reveal that N-bromoacetyldistamycin binds within minutes to a 167 base pair (bp) restriction fragment at four A,T rich sites five base pairs in size, 5'-TTTAA, GTTTA, AAATT, and GAAAT-3'. After a reaction time of 5 h at 37 °C, cleavage occurs at a single adenine in the GTTTA site in this 167 bp restriction fragment following a piperidine workup procedure. Covalent attachment of BD occurs at N3 of adenine. 3-(Acetyldistamycin)adenine is the released product from the reaction of BD with a 15 base pair oligonucleotide duplex 5'-CGGTAGTTTATCACA-3' at 37 °C. The 3' and 5' DNA termini at the site of cleavage after piperidine treatment are phosphate groups. Alkylation of adenine in duplex DNA by BD is described by a first-order rate process. The rate constant for alkylation of N3 of adenine at the sequence 5'-GTTTA-3' is k = 5.39 X 10^(-6)s^(-1) (t_(1/2) = 35.7 h) at 37 °C. The cleavage specificities of the synthetic molecule, N-bromoacetyldistamycin, and the natural product, CC-1065, were compared on the 167 bp restriction fragment. After a reaction time of 1 h at 37 °C, CC-1065 cleaves at 13 adenines whereas BD shows cleavage at one adenine. The differences in covalent binding specificity between CC-1065 and BD may be related to sequence dependent reactivity in both cases but sequence dependent binding being additionally important in the latter.