Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates

SIMPLE SUMMARY: During replication, DNA molecules undergo topological changes that affect supercoiling, catenation and knotting. To better understand this process and the role of topoisomerases, the enzymes that control DNA topology in in vivo, two-dimensional agarose gel electrophoresis were used to investigate the efficiency of three type II DNA topoisomerases, the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α, on partially replicated bacterial plasmids containing replication forks stalled at specific sites. The results obtained revealed that despite the fact these DNA topoisomerases may have evolved to accomplish specific tasks, they share abilities. To our knowledge, this is the first time two-dimensional agarose gel electrophoresis have been used to examine the ability of these topoisomerases to relax supercoiling in the un-replicated region and unlink pre-catenanes in the replicated one of partially replicated molecules in vitro. The methodology described here can be used to study the role of different topoisomerases in partially replicated molecules. ABSTRACT: DNA topoisomerases are the enzymes that regulate DNA topology in all living cells. Since the discovery and purification of ω (omega), when the first were topoisomerase identified, the function of many topoisomerases has been examined. However, their ability to relax supercoiling and unlink the pre-catenanes of partially replicated molecules has received little attention. Here, we used two-dimensional agarose gel electrophoresis to test the function of three type II DNA topoisomerases in vitro: the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α. We examined the proficiency of these topoisomerases on a partially replicated bacterial plasmid: pBR-TerE@AatII, with an unidirectional replicating fork, stalled when approximately half of the plasmid had been replicated in vivo. DNA was isolated from two strains of Escherichia coli: DH5αF’ and parE10. These experiments allowed us to assess, for the first time, the efficiency of the topoisomerases examined to resolve supercoiling and pre-catenanes in partially replicated molecules and fully replicated catenanes formed in vivo. The results obtained revealed the preferential functions and also some redundancy in the abilities of these DNA topoisomerases in vitro.