Dynamic Investigation of DNA Bending and Wrapping By Type II Topoisomerases

Abstract

Type II topoisomerases catalyze DNA decatenation and unwinding which is crucial for cell division, and therefore type II topoisomerases are some of the main targets of anti-cancer drugs. A recent crystal structure shows that, during the catalytic cycle, a yeast type II topoimerase can bend a 34 base pair DNA segment by up to 150 degrees. Bacterial gyrase, another type II topoisomerase, can wrap an approximately 100 bp DNA segment into a tight 180 degree turn. Bending a stiff polymer like DNA requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. By substitututing diaminopurine (DAP) deoxyribonucleotides for dATP in PCR reactions, stiffer DNA fragments have been produced and used as substrates for topoisomerase II-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. The overall rate of relaxation of plectonemes by recombinant human topoisomerase II alpha decreased on the stiffer DNA. In addition the ability of recombinant E. coli gyrase to wrap DNA also decreased for DAP-substituted DNA in which every base pair has three hydrogen bonds. These dynamic measurements of DNA bending and wrapping by type II topisomerases are consistent with the hypothesis that DNA flexibility affects the rate determining step for type II topoisomerase activity.