Control of gyration and reverse gyration machineries – CtrlAltGyr

DNA topoisomerases are essential enzymes regulating in DNA topology during DNA replication, transcription, repair, recombination, chromosome segregation and genome stability. If most topoisomerases relax supercoiled DNA, gyrase and the reverse gyrase, are uniquely able to introduce supercoils into DNA. DNA gyrase is present in all bacteria and in mesophilic archaea, and reverse gyrase in all hyperthermophilic organisms. DNA gyrase cleaves transiently two DNA strands to introduce negative supercoils while reverse gyrase cleaves only one DNA strand to produce positive supercoils.
Despite their mechanistic difference, these topoisomerases share a CAP domain harboring the universally conserved catalytic tyrosine, and a TOPRIM domain that plays an important role in the transesterification reaction. In both gyrase and reverse gyrase, these two essential domains are associated with a different ATPase motor, a GHKL or SF2 helicase domain respectively. ATP hydrolysis by these two nanomachines leads to a high energetic state of DNA by transporting one DNA molecule through another DNA transient break in one direction. The main objective of this proposal is to elucidate how these molecular machines transport DNA in only one direction to produce a DNA in a high energy state of opposite polarity using a distinct combination of structural domains. Despite extensive analysis of the gyration and reverse gyration mechanisms, the correlation between particular catalytic steps recorded in kinetic studies related to discreet conformational states remains largely a matter of speculation. This correlation is limited by the availability of full length 3D structures representing different conformational states of the nucleoprotein complexes on relevant DNA templates. Answering this question requires the analysis of the architecture of gyrase and reverse gyrase complexes on a DNA template with defined and controlled topology.
To achieve this goal, the kinetic and the interaction of their complex with DNA molecules need to be controlled to freeze the most favorable conformation. Using magnetic tweezers, we will perform single molecule kinetics measurements to determine the kinetic stability of the nucleoprotein complexes and monitor the composition of the complex that will lead to the most stable state when adding small molecules. Using single molecule cryo-electron microscopy (CryoEM), we will analyze the 3D architecture of a stable conformation of gyrase on DNA crossovers and reverse gyrase locked on double-stranded DNA. The use of different ATP analogs and drugs targeting gyrase or the reverse gyrase TopoIA domain, will help us to trap particular states of the catalytic cycle. We will combine kinetics measurements with structural analysis of the conformations of both enzymes using cutting-edge single molecule magnetic tweezers and cryo-electron microscopy approaches. Magnetic tweezers allow to quantify in real time the conformational changes that occur into a single DNA molecule while, by analyzing a large set of single particles, cryo-electron microscopy allow to analyse the different conformations and conformational changes of the topoisomerases in interaction with DNA. The thermophilic nature of the enzymes that we will use in this proposal will facilitate sample preparation and data acquisition of discreet states leading to tridimensional reconstruction of the two enzymes on DNA. Complementary kinetic data will allow us to understand the relation between the ATP motor and supercoiling reaction. All these data, obtained with complementary approaches will give us an integrated dynamic view of the gyration and reverse gyration processes.