Fast dynamics of supercoiled DNA revealed by single-molecule experiments

Abstract

The dynamics of supercoiled DNA play an important role in various cellular processes such as transcription and replication that involve DNA supercoiling. We present experiments that enhance our understanding of these dynamics by measuring the intrinsic response of single DNA molecules to sudden changes in tension or torsion. The observed dynamics can be accurately described by quasistatic models, independent of the degree of supercoiling initially present in the molecules. In particular, the dynamics are not affected by the continuous removal of the plectonemes. These results set an upper bound on the hydrodynamic drag opposing plectoneme removal, and thus provide a quantitative baseline for the dynamics of bare DNA.magnetic tweezers ͉ optical tweezers ͉ polymer dynamics T he degree of DNA supercoiling affects a number of important cellular processes, such as gene expression (1), initiation of DNA replication (2), binding kinetics of sequence-specific proteins to their targets (3), and site-specific recombination (4, 5). A strict regulation of DNA supercoiling is therefore essential for cell survival. This regulation results from a complex interplay between the occurrence of processes that generate local supercoiling of DNA, such as replication and transcription, and the action of topoisomerases, which are able to modify the global linking number (Lk) of DNA molecules via a mechanism of transient DNA strand breakage and religation (for reviews, see refs. 6 and 7).DNA supercoiling dynamics, i.e., the rate at which supercoils are created, propagated and removed on a DNA molecule, represent an important aspect of the regulation process. This can be clearly illustrated by the example of transcription-induced supercoiling. As initially proposed by Liu and Wang (8) and confirmed by later experiments in vitro (9) and in vivo (10, 11), the inability of a transcription complex of increasing molecular weight to rotate around helical DNA results in the supercoiling of DNA in its immediate vicinity: positively supercoiled domains are generated ahead of the transcription complex, whereas negatively supercoiled domains are generated behind it. In the simple case of a single transcription complex bound to a circular DNA molecule, these supercoiled domains of opposite sign can be relaxed in one of two ways, either by the action of topoisomerases or by their mutual annihilation following their propagation along the connecting DNA segment. These two processes can have very different consequences for DNA topology: the action of topoisomerases induces a modification of Lk unless these enzymes relax positive and negative supercoils in a perfectly balanced way, whereas the merging of oppositely supercoiled domains does not influence the global Lk. Thus, the relative kinetics of these two processes appears as a major determinant of the degree of supercoiling of DNA in steady state. Within this context, DNA internal dynamics play an important role because they determine the rate at which oppositely supercoiled domains propagate and mer...

Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end

“…For the experiments of this paper we estimate such volume-exclusion forces to be well below 0.1 pN and therefore irrelevant. We also note that for relaxation of supercoiling there is an additional contribution to the drag coefficient during relaxation from rotation of the plectonemic domain; however, a detailed study of this reached the conclusion that plectoneme drag was a negligible contribution to the total drag coefficient opposing relaxation (15) for topo IB relaxation experiments similar to those reported here.…”

Dynamic analysis of a diffusing particle in a trapping potential

“…Few aspects of DNA dynamics have been treated before, such as the DNA dynamics in a strong stretching regime where r ∼ L [17], the DNA dynamics in response to stretching forces [18], or the dynamics of a tethered particle in the case of DNA looping [19,20] [22,23], and the statistics of Markovian processes for measuring the rate of loop formation due to protein action [19,20]. The full dynamics as we described above, however, have not been considered.…”

Dynamics of phosphodiester synthesis by DNA ligase

“…For instance, Ͼ80% of the intermediate steps observed under F ϭ 0.5 pN were accurately described by the quasistatic model used in ref. 28 (Fig. 4a and SI Text), which takes into account the magnetic force, the drag opposing the motion of the bead linked to DNA, and the tension in the DNA, where for the latter we assume the equilibrium force-extension relation at the degree of supercoiling present in the DNA at the end of the relaxation.…”