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• W2006076913 abstract "A recent study has analysed the action of bacterial DNA gyrase on a single substrate DNA molecule, discriminating the initial DNA wrapping and subsequent supercoiling steps in the reaction cycle. A recent study has analysed the action of bacterial DNA gyrase on a single substrate DNA molecule, discriminating the initial DNA wrapping and subsequent supercoiling steps in the reaction cycle. One of the most intriguing developments of the last few years has been the advent of methodology for investigating biopolymer molecules and enzyme action at the single molecule level [1Wallace M.I. Malloy J.E. Trentham D.R. Combined single-molecule force and fluorescence measurements for biology.J. Biol. 2003; 2: 4Crossref PubMed Google Scholar, 2Charvin G. Strick T.R. Bensimon D. Croquette V. Tracking topoisomerase activity at the single-molecule level.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 201-219Crossref PubMed Scopus (88) Google Scholar]. This fascinating technology has the advantage that the details of enzymatic events may, in principle, be investigated free of the averaging problems of bulk, ensemble measurements, and those associated with the presence of an unknown fraction of inactive or defective enzyme in the sample. But care must be taken in interpreting the results, as the experimental setups often do not mimic the bulk solution conditions in important respects. This technology has been applied to the analysis of a number of topoisomerases [2Charvin G. Strick T.R. Bensimon D. Croquette V. Tracking topoisomerase activity at the single-molecule level.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 201-219Crossref PubMed Scopus (88) Google Scholar] and has now been used by Gore et al. [3Gore J. Bryant Z. Stone M.D. Nöllmann M. Cozzarelli N.R. Bustamante C. Mechanochemical analysis of DNA gyrase using rotor bead tracking.Nature. 2006; 439: 100-104Crossref PubMed Scopus (163) Google Scholar] to investigate the activity of the archetypal type II topoisomerase, bacterial DNA gyrase. DNA topoisomerases manipulate the topological forms of DNA: supercoiled, catenated (linked) and knotted molecules [4Champoux J.J. DNA topoisomerases: structure, function, and mechanism.Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2180) Google Scholar, 5Bates A.D. Maxwell A. DNA Topology. Oxford University Press, Oxford2005Google Scholar]. Type II topoisomerases are molecular machines that capture a DNA double strand, the T (transported) segment, in a clamp formed by ATP-dependent dimerisation of the amino-terminal domains of the dimeric enzyme. The T segment is passed through a second DNA, the G (gate) segment, via a double-stranded break formed by phosphodiester exchange with tyrosine residues in the protein. An intramolecular ‘strand-passage’ reaction can result in relaxation of supercoils — the linking number of closed-circular DNA changes by ± 2 — or knotting/unknotting of DNA. Catenation or decatenation occurs if the G and T segments are on separate molecules. All type II topoisomerases carry out these reactions, but DNA gyrase has evolved specifically to transduce the free energy of ATP hydrolysis into negative supercoiling (Figure 1). The enzyme wraps approximately 130 base pairs of DNA, including the G segment, in a right-handed sense around the carboxy-terminal domains of the GyrA subunits, to present a contiguous T segment to the ATP-operated clamp with an orientation leading to a change in linking number of –2. The experimental setup designed to analyse the activity of a single gyrase enzyme is illustrated in Figure 2A [3Gore J. Bryant Z. Stone M.D. Nöllmann M. Cozzarelli N.R. Bustamante C. Mechanochemical analysis of DNA gyrase using rotor bead tracking.Nature. 2006; 439: 100-104Crossref PubMed Scopus (163) Google Scholar, 6Bryant Z. Stone M.D. Gore J. Smith S.B. Cozzarelli N.R. Bustamante C. Structural transitions and elasticity from torque measurements on DNA.Nature. 2003; 424: 338-341Crossref PubMed Scopus (458) Google Scholar]. An approximately 10 kilobase DNA molecule (3 μm) is stretched between a coverslip and a magnetic bead, and a force of a up to 2 piconewtons (pN) is applied magnetically. The double-stranded DNA has a single nick in one strand, immediately below which is attached a smaller fluorescent bead. Gyrase action at a specific binding sequence below the bead introduces negative supercoils, which, since the DNA is under tension, are manifested as untwisting of the DNA. This untwisting is relaxed by rotation at the DNA nick and concomitant rotation of the marker bead is observed from below using a fluorescence microscope. Figure 2B shows schematic plots of bead rotation angle against time, for a series of tensile forces applied to the DNA [3Gore J. Bryant Z. Stone M.D. Nöllmann M. Cozzarelli N.R. Bustamante C. Mechanochemical analysis of DNA gyrase using rotor bead tracking.Nature. 2006; 439: 100-104Crossref PubMed Scopus (163) Google Scholar]. These plots show bursts of rotation of the marker bead, always in multiples of two rotations in the same direction, consistent with the introduction of two negative supercoils per gyrase reaction. Interestingly, as the force applied to the DNA is increased, the size and frequency of the bursts — corresponding to multiple processive gyrase reactions — decreases, but the rate of rotation is force-independent. These data are interpreted in terms of a model in which the initiation and maintenance of a processive burst depends on a competition between DNA dissociation and DNA wrapping of an unwrapped intermediate [3Gore J. Bryant Z. Stone M.D. Nöllmann M. Cozzarelli N.R. Bustamante C. Mechanochemical analysis of DNA gyrase using rotor bead tracking.Nature. 2006; 439: 100-104Crossref PubMed Scopus (163) Google Scholar]. The wrapping step leads to a shortening of the DNA, and hence will slow down as tension in the DNA increases. Quantitative analysis suggests that DNA shortening at the wrapping transition state is about 31 nm (90 base pairs), a value consistent with a full wrap of approximately 130 base pairs (the transition state may be only partially wrapped). On the other hand, the fact that the rate of the processive reaction is independent of the force suggests that the rate-limiting step of the reaction does not involve wrapping (or unwrapping) of the DNA. The measured rate is very consistent with the known rate of ATP hydrolysis by gyrase in the presence of DNA [7Bates A.D. O'Dea M.H. Gellert M. Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage and DNA supercoiling.Biochemistry. 1996; 35: 1408-1416Crossref PubMed Scopus (53) Google Scholar, 8Reece R.J. Maxwell A. DNA gyrase: Structure, mechanism, and interaction with antibiotics.CRC Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (557) Google Scholar]. In a variant of the assay designed to have better time resolution, pauses are visible during the processive reaction bursts, corresponding to complete two-rotation steps, or rarely to steps corresponding to about one rotation of the bead. To explain the pauses, Gore et al. [3Gore J. Bryant Z. Stone M.D. Nöllmann M. Cozzarelli N.R. Bustamante C. Mechanochemical analysis of DNA gyrase using rotor bead tracking.Nature. 2006; 439: 100-104Crossref PubMed Scopus (163) Google Scholar] propose an undefined rate-limiting step late in the reaction cycle. However, post-hydrolysis ADP release has previously been proposed as the rate-limiting step [9Ali J.A. Jackson A.P. Howells A.J. Maxwell A. The 43 kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyses ATP and binds coumarin drugs.Biochemistry. 1993; 32: 2717-2724Crossref PubMed Scopus (310) Google Scholar, 10Ali J.A. Orphanides G. Maxwell A. Nucleotide binding to the 43-kilodalton N-terminal fragment of the DNA gyrase B protein.Biochemistry. 1995; 34: 9801-9808Crossref PubMed Scopus (90) Google Scholar], and this is consistent with the present data (Figure 3). At low or zero ATP concentration, the one-rotation steps are more common, and reversible, and are suggested to correspond to formation of the wrapped complex (Figure 1) before (or without) strand passage. The exact rotation/wrap measured (1.3 turns) is rather larger than the value expected from well-established average bulk measurements of 0.7–0.8 turns [8Reece R.J. Maxwell A. DNA gyrase: Structure, mechanism, and interaction with antibiotics.CRC Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (557) Google Scholar, 11Kampranis S.C. Bates A.D. Maxwell A. A model for the mechanism of strand-passage by DNA gyrase.Proc. Natl. Acad. Sci. USA. 1999; 96: 8414-8419Crossref PubMed Scopus (126) Google Scholar, 12Heddle J.G. Mitelheiser S. Maxwell A. Thomson N.H. Nucleotide binding to DNA gyrase causes loss of the DNA wrap.J. Mol. Biol. 2004; 337: 597-610Crossref PubMed Scopus (63) Google Scholar]; either a proportion of the enzyme is defective in wrapping, or the extent of wrapping is dependent on the DNA binding site and this very specific site has a larger than average wrap. Gore et al. [3Gore J. Bryant Z. Stone M.D. Nöllmann M. Cozzarelli N.R. Bustamante C. Mechanochemical analysis of DNA gyrase using rotor bead tracking.Nature. 2006; 439: 100-104Crossref PubMed Scopus (163) Google Scholar] propose a model of gyrase action that explains their observations. However, there are some features of this model that are inconsistent with existing biochemical data relating to gyrase mechanism; in particular, hydrolysis product release as the rate limiting step (see above), and variation in the efficiency of coupling between ATP binding and strand passage. A modified scheme that aims to reconcile the single molecule data with previous work is proposed in Figure 3: the ATP-dependent supercoiling reaction under normal conditions is the square (2–5), where it is supposed that the enzyme can cycle without the DNA becoming unwrapped from the carboxy-terminal domains. Gore et al. [3Gore J. Bryant Z. Stone M.D. Nöllmann M. Cozzarelli N.R. Bustamante C. Mechanochemical analysis of DNA gyrase using rotor bead tracking.Nature. 2006; 439: 100-104Crossref PubMed Scopus (163) Google Scholar] state that when ATP is saturating, wrapping of the DNA in the gyrase complex leads inevitably to supercoiling, suggesting that subsequent rapid binding of ATP commits the enzyme to the strand-passage reaction. This seems to be the case in their experiments, but there is considerable earlier evidence that this coupling is not perfect; ATP binding and hydrolysis can take place without strand passage [7Bates A.D. O'Dea M.H. Gellert M. Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage and DNA supercoiling.Biochemistry. 1996; 35: 1408-1416Crossref PubMed Scopus (53) Google Scholar, 13Sugino A. Higgins N.P. Brown P.O. Peebles C.L. Cozzarelli N.R. Energy coupling in DNA gyrase and the mechanism of action of novobiocin.Proc. Natl. Acad. Sci. USA. 1978; 75: 4838-4842Crossref PubMed Scopus (327) Google Scholar]. Activation of ATP hydrolysis requires the presence of a T segment in the clamp [14Maxwell A. Gellert M. The DNA dependence of the ATPase activity of DNA gyrase.J. Biol. Chem. 1984; 259: 14472-14480Abstract Full Text PDF PubMed Google Scholar, 15Tingey A. Maxwell A. Probing the role of the ATP-operated clamp in the strand-passage reaction of DNA gyrase.Nucl. Acids Res. 1996; 24: 4868-4873Crossref PubMed Scopus (75) Google Scholar], which should always be captured, but may be released after ATP hydrolysis, without strand passage (cycling between 2 and 3 in Figure 3) [11Kampranis S.C. Bates A.D. Maxwell A. A model for the mechanism of strand-passage by DNA gyrase.Proc. Natl. Acad. Sci. USA. 1999; 96: 8414-8419Crossref PubMed Scopus (126) Google Scholar]. The probability of strand passage is dependent on the level of supercoiling of the substrate DNA, declining as the DNA becomes more negatively supercoiled [7Bates A.D. O'Dea M.H. Gellert M. Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage and DNA supercoiling.Biochemistry. 1996; 35: 1408-1416Crossref PubMed Scopus (53) Google Scholar]; it has been proposed that supercoiling torsion affects the position of a strand passage equilibrium (step 3–4) [11Kampranis S.C. Bates A.D. Maxwell A. A model for the mechanism of strand-passage by DNA gyrase.Proc. Natl. Acad. Sci. USA. 1999; 96: 8414-8419Crossref PubMed Scopus (126) Google Scholar]. The coupling efficiency is only around 30% for relaxed DNA [7Bates A.D. O'Dea M.H. Gellert M. Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage and DNA supercoiling.Biochemistry. 1996; 35: 1408-1416Crossref PubMed Scopus (53) Google Scholar]. The fact that strand passage has an apparently high probability in these experiments — although some of the rare single-turn pauses may represent uncoupled reactions — is plausibly due to the fact that the DNA is under tension. The tensile force will directly affect the wrapping/unwrapping equilibria in Figure 3, making the putative unwrapped intermediates 6 and 7 more significant, with a force-dependent increase in the rate of dissociation of the enzyme–DNA complex (from 7 or 2) and shorter, less frequent bursts of activity. In this scheme, the product release step is rate-limiting independent of force. The equilibria in Figure 3 would be perturbed by tension as indicated by the red arrows, with the strand passage step being increased in efficiency, leading to the high level of coupling between ATP binding and supercoiling seen in the single-molecule experiments, but not in analogous bulk experiments. These experiments have revealed an interesting snapshot of the operation of single gyrase enzymes, although it is important to try to reconcile the new data with previous bulk experiments. In this context, it would be interesting to analyse the enzyme's activity at lower levels of tensile force, perhaps using methods where the DNA forms writhed, plectonemic supercoils [2Charvin G. Strick T.R. Bensimon D. Croquette V. Tracking topoisomerase activity at the single-molecule level.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 201-219Crossref PubMed Scopus (88) Google Scholar], in order to investigate the uncoupling of ATP binding and strand-passage. More speculatively, it would be fascinating to have a corresponding single-molecule method to analyse ATP hydrolysis simultaneously with DNA supercoiling, to really dissect the energy coupling aspects of these systems." @default.
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• W2006076913 date "2006-03-01" @default.
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• W2006076913 title "DNA Topoisomerases: Single Gyrase Caught in the Act" @default.
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