Book of Abstracts: Albany 2005
Duplex-passage Reaction Between Short Double-stranded DNA Regions Within Long Circular Single-stranded DNA Mediated by Type II Topoisomerases
The fundamental property of type II topoisomerases (topo II) is their ability to transfer one DNA double-stranded region through another by creating and rejoining double-stranded DNA break in one of the reacting DNA duplexes. This reaction can lead to DNA catenation/decatenation, knotting/unknotting or the introduction/removal of DNA supercoils. These reactions play important biological roles, for example in DNA replication. To elucidate minimal requirements for this reaction we investigated the duplex-passage mediated by type II topos between short regions of double-stranded DNA obtained by annealing of single-stranded oligonucleotides to the long circular single-stranded DNA. This reaction leads to catenation of DNA circles. To facilitate catenation we bring substrate DNA circles together using tethering oligonucleotides, which are removed after reaction is completed. This system allows to vary the length of the duplexes, distances between reacting duplexes and to use various DNA analogs as topo II substrates. Using this system we show that in the case of E. coli DNA gyrase and yeast topo II, DNA duplex regions as short as 20 bp are sufficient for catenation, and for DNA gyrase at least one of the duplex regions could be as short as 12 bp.
Boris P. Belotserkovskii
Department of Molecular and Cell biology