DNA linking potential generated by gyrase

Abstract

Whether or not DNA gyrase can supercoil DNA so that alternative structures will arise in it is the major question of this work. We have shown gyrase to produce in pA03 DNA a superhelix density sufficient for cruciform formation. However, the transition does not take place because of too slow kinetics. A change of ionic conditions in favour of more intense DNA supercoiling by gyrase shifts the midpoint of the equilibrium transition to the cruciform structure toward more supercoiled topoisomers.The width of the equilibrium transition to the cruciform as a function of linking number has been revealed to be an order of magnitude larger in buffers containing magnesium and spermidine than in buffers with monovalent cations only. We ascribe this effect to the influence that the counter ions surrounding the DNA molecule have on its elasticity, the coefficient of elasticity being dependent on superhelix density u. Thus, the free energy of supercoiling (a) depends on the ionic conditions and (b) is not a quadratic function of u in the physiological range of parameters.We propose a description of DNA as a system of links that can be either closed or open; we also introduce a new concept of the DNA linking potential akin to the chemical and electric potentials. The linking potential is a suitable parameter for describing the equilibrium distribution of links in heterogeneous DNA, the coexistence of various DNA structures, the equilibrium input and output of DNA links by enzymes, and the nonequilibrium movement of links along DNA chains. Within the framework of this approach DNA gyrase is considered as the source of the DNA linking potential.The superhelical stress in cellular DNA can be permanent; it can also arise during transcription, replication, homologous recombination [I -81. DNA superhelicity in vivo is controlled by such enzymes as DNA topoisomerases, polymerases, helicases, Rec A [2 -121. An approach has been developed for the investigation of negative superhelicity in DNA in vivo that consists in assaying the DNA structural transition in vivo followed by a comparison with the scaling measurements in vitro [13-251. The advantages and limitations of this approach have been discussed [22, 231. Previously we have performed comparative studies of cruciformation in pA03 DNA in vivo and in vitro [26-301 and obtained the following results. The cruciform was absent from intracellular pA03, although after DNA was extracted the transition to the cruciform occurred in these topoisomers during incubation in a 20 mM sodium buffer [29]. A possible Definitions. N , DNA length in bp; Lk, linking number; Lko, linking number of a relaxed DNA; 0, specific linking difference, or superhelix density, 0 = (Lk-Lko)/Lko; @, linking density, Q = Lk/ N ; GSh, free energy of a superhelical DNA; G', free energy of cruciformation; GGyr, effective free energy transduced to DNA by gyrase in one cycle; R, the gas constant; p + , probability of cruciformation; L k + , midpoint of cruciformation, Lk at p + = 1/2; A', width of cruciformation, A' = ...

Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements

“…Gyrase, too, is far more active on (+) SC DNA than on relaxed or (−) SC DNA (Westerhoff et al 1988;Kozyavkin et al 1990). However, substrate discrimination by gyrase is due to a chiral wrapping of 135 bp of DNA around the enzyme that is favored by (+) supercoiling (Morrison et al 1980;Kirkegaard and Wang 1981).…”

“…In addition to our data here on topo IV, there are a number of experiments that show that DNA gyrase is much more active on (+) than (−) supercoils. Its activity is reduced by 10-fold at = −0.06 compared with relaxed DNA (Westerhoff et al 1988;Kozyavkin et al 1990). Given the essential role of (−) supercoiling, we suppose that there is a patch of (+) supercoiling around the relication fork that is maintained by topological domain barriers.…”

Rgen-Seq for Highly Sensitive Amplification-Free Screen of Off-Target Sites of Gene Editors

“…By contrast, in CIRCLE-seq and CHANGE-seq closed circular DNA of different sizes gets cleaved by Cas9. Depending on circularization conditions such as, for example, the ~20°C temperature difference between the circularization and Cas9 cleavage reactions 26,27 , closed circular molecules can be under the helix stabilizing torsional stress and even be partially positively supercoiled at the cleavage conditions, depending on the DNA size [41][42][43] . Such torsional stress and associated positive supercoiling energy can impose a significant barrier for the Cas9:sgRNA-medited R-loop formation 44,45 , resulting in negative selection of off-target sites with lower affinity to Cas9:sgRNA complexes.…”

RGEN-seq For Highly Sensitive Amplification-free Screen Of Off-target Sites Of Gene Editors

“…By contrast, in CIRCLE-seq and CHANGE-seq closed circular DNA of different sizes gets cleaved by Cas9. Depending on circularization conditions such as, for example, the ~20°C temperature difference between the circularization and Cas9 cleavage reactions 26,27 , closed circular molecules can be under the helix stabilizing torsional stress and even be partially positively supercoiled at the cleavage conditions, depending on the DNA size [42][43][44] . Such torsional stress and associated positive supercoiling energy can impose a signi cant barrier for the Cas9:sgRNA-medited R-loop formation 45,46 , resulting in negative selection of off-target sites with lower a nity to Cas9:sgRNA complexes.…”