Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by flp recombinase

Abstract

Hydrogen peroxide is capable of effecting the cleavage of a specific phosphodiester bond in DNA, when used in concert with the recombinase enzyme Flp from Saccharomyces cerevisiae. This cleavage is not caused by oxidative damage of the DNA backbone but instead is the result of nucleophilic attack by peroxide. A single phosphorus-oxygen bond is broken in the reaction. Cleavage of DNA by peroxide also occurs with an inactive mutant of Flp in which the active site nucleophile tyrosine has been replaced by phenylalanine. Besides providing information on the mechanism of strand cleavage by Flp, these results may contribute to the development of new synthetic DNA cleavage reagents that act by hydrolytic and not radical chemistry.