Using 3'-bridging phosphorothiolates to isolate the forward DNA cleavage reaction of human topoisomerase IIalpha.

ABSTRACT: The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3'-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3'-terminal -SH moiety that is a poor nucleophile relative to the normal 3'-terminal -OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomer

SUBMITTER: Deweese JE 

PROVIDER: S-EPMC2733888 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature