Abstract
Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair. While the catalytic cycle of Type 1A topoisomerases has been long-known to involve an enzyme-bridged ssDNA gate that allows strand passage, a deeper mechanistic understanding of these enzymes has only recently begun to emerge. This knowledge has been greatly enhanced through the combination of biochemical studies and increasingly sophisticated single-molecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy and Förster resonance energy transfer. In this review, we discuss how single-molecule assays have advanced our understanding of the gate opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the interplay of Type 1A topoisomerases with partner proteins, such as RecQ-family helicases. We also highlight how these assays have shed new light on the likely functional roles of Type 1A topoisomerases in vivo and discuss recent developments in single-molecule technologies that could be applied to further enhance our understanding of these essential enzymes.
Highlights
Many genomic processes, including DNA replication, transcription and repair, result in torsional stress being exerted on DNA
We explore how our mechanistic understanding of these vital enzymes has advanced greatly in recent years through the application of singlemolecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy (AFM) and single-molecule Forster resonance energy transfer
As we have discussed in detail in this review, the application of single-molecule assays has yielded significant new insights into the molecular mechanisms and reaction kinetics of Type 1A topoisomerases, as well as the mechanistic interactions of these enzymes with partner proteins
Summary
Many genomic processes, including DNA replication, transcription and repair, result in torsional stress being exerted on DNA. These studies exploit the ability of magnetic tweezers to apply both torsional stress and tension to single molecules of DNA and have revealed a significant influence of the DNA tension on the ability of Type 1A topoisomerases to relax supercoils This observation is, in part, due to the fact that negatively supercoiled DNA can adopt different structures depending on the applied tension (Figure 4A). By measuring the change in DNA end-to-end length at constant force (through monitoring the bead height), magnetic tweezers studies have demonstrated that, on negatively supercoiled DNA, Type 1A topoisomerases show substantially lower relaxation activity at forces
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