KINETIC-ANALYSIS OF DRUG CLEAVAGE OF CLOSED-CIRCULAR DNA

Various cleavage agents interact with circular double-stranded DNA mol ecules to convert the closed-circular form (form-I) to the open-circul ar (form-II) and linear (form-III) forms, and ultimately to small DNA fragments. The various cutting processes which take place in the DNA p ool are here analyzed kinetically, and, by solving the kinetic equatio ns, expressions are derived for the amounts of the closed-circular, op en-circular, and linear forms of DNA as a function of reaction time an d concentration of cleavage agent. Conversions between subspecies of f orms II and III, differing in numbers of internal cuts, are taken into account. The only assumption required to solve the kinetic equations is that the concentration of cleavage agent obeys [D] = D-0 f(t) where the function of time f(t) is independent of D-0, the initial concentr ation of cleavage agent. By choosing parameters in the expressions for the calculated amounts of forms I, II and III to give the best fit to the measured amounts, one obtains information about the rates and rat e constants for the conversions. The rate constants in turn give impor tant information about the specificity and mechanism of action of the cleavage agents. The analysis is applied to the cleavage of pBR322, SV -40 and PM2 DNAs by DNase I, Fe-EDTA, and the antitumor agents caliche amicin and bleomycin. Cleavage rate constants are derived and discusse d for these systems. (C) 1997 Elsevier Science B.V.