By circularizing short DNA molecules that differed in their length by a fraction of one helical turn, we obtained DNA minicircles with varying levels of stored torsional energy. DNA minicircles produced with 106
bp (MC106) and 108
bp (MC108) were designed to be less untwisted than their 100-bp counterpart (MC100). In an attempt to assay for the presence of disrupted DNA structures in these minicircles, we incubated each minicircle in the presence of an enzyme (Bal31 nuclease) that is known to specifically digest non-canonical DNA structures. Owing to their resistance to degradation by Bal31 nuclease, we concluded that MC106 and MC108 did not contain torsionally destabilized DNA regions that would be sensitive to Bal31 nuclease. Using cryo-EM imaging in combination with 3D reconstruction, these minicircles were shown to adopt nearly circular shapes. We have further shown through Brownian dynamics simulations that the small levels of ellipticity that were observed in MC106 and MC108 are well within the range that is expected for thermally fluctuating DNA minicircles of this size, even if they are torsionally stressed. In sharp contrast to the less underwound DNA minicircles, MC100 molecules were underwound by nearly half of a helical turn. It is important to note that such a level of underwinding is likely to be higher than that typically maintained in the cell on a global level. However, it has been shown that the degree to which DNA can be locally underwound can be transiently higher than global levels (47
). Since locally enhanced levels of torsional stress can induce the formation of non-canonical DNA structures (6
), MC100 can be used as a model system to study these stress-induced structures under conditions that may be transiently present in vivo
. As demonstrated here, the elevated level of negative torsional stress in MC100 resulted in the formation of kinked regions that were sensitive to Bal31 nuclease. Based in part on results from previous work characterizing the helical pitch of kinked, untwisted DNA (15
), we estimated that disruptions to the helical structure of MC100 should not involve more than two dinucleotide steps due to passing from a right-handed helix with about 10.5
bp/turn to a left-handed arrangement with ~13
bp/turn, the extent of underwinding will be reduced by 62° for each affected dinucleotide stack. As discussed previously, the untwisted MC100 has been estimated to sustain a total twist deficit of 173°. A switch of two dinucleotide stacks from right- to left-handed geometry will reduce the twist deficit to 49°, which would be sufficient to ensure that the twisting angle in the remaining portion of right-handed DNA remains below the critical underwinding angle of 0.6°/bp (15
). We observed using cryo-EM that 100-bp DNA minicircles adopted a strongly elliptical shape that is consistent with a model in which kinked sites in DNA are highly flexible to bending. Since MC100 is the smallest of the three minicircles tested, one possible explanation accounting for the appearance of stress-induced, highly flexible sites in MC100 is that the bending energy sustained within the minicircle is sufficient to precipitate the transition into an alternative DNA conformation. However, although MC100 sustains higher magnitudes of bending stress than MC106 and MC108, it was shown by Du et al.
) that 85-bp minicircles do not undergo inelastic kinking due to bending alone. Therefore, it is highly unlikely that the flexible sites observed in MC100 arise simply due to bending alone. We expect that kinking occurs preferentially at sites that are easily meltable; however, the resolution of cryo-EM does not permit us to relate the DNA sequence to observed sites of kinks.
To provide support for our conclusion that kinked regions are highly flexible to bending, we performed Brownian dynamics simulations of DNA minicircles. Our numerical model predicts the 3D shape of DNA minicircles under thermal energy alone, and considers cases where the DNA structure was either uniformly elastic (as with canonical DNA) or contains one or more hyperflexible sites (as we expect for DNA containing localized, kinks). To our surprise, simulations revealed that under the conditions present during cryo-EM, one highly flexible site is not sufficient to permit the modeled DNA minicircles to adopt the degree of ellipticity observed for MC100 (Supplementary Figure S1
). Influenced by recent atomistic simulations demonstrating the formation of two kinks in underwound DNA minicircles (10
), we performed additional Brownian dynamics simulations of DNA minicircles containing two highly flexible sites that are located at opposite points along the circumference of the minicircle. Indeed, the modeled minicircles with two highly flexible sites adopted shapes with similar ellipticities to MC100. Our simulations suggest that the creation of a first hyperflexible site in a DNA minicircle induces high curvature within the region opposite the initial kink, triggering the formation of a second kink in a cooperative manner.
presents our model for structural cooperativity during kinking at distant sites in small DNA loops (also, see Movie S1 in Supplementary Data
). Here, we illustrate a 100-bp DNA minicircle that is simulated by a numerical, elastic rod model. As previously discussed, a minicircle of this size is strongly bent but does not kink due to bending alone. As negative torsional stress is increased in this highly bent minicircle, the helical structure of DNA locally destabilizes and a kink is formed (A–C). As the bending energy within the entire minicircle equilibrates, the unkinked portion of the DNA adopts a teardrop-like conformation in which the local bending curvature nearest to the kink is reduced and gradually increases as a function of contour distance from the kinked site (D). Although the formation of a first kink reduces the overall bending energy within the minicircle, the resulting distribution of bending curvatures localizes the maximal bending stresses in the region of the minicircle farthest from the first kink (E). This concentrated, local bending stress, in turn, provides an energetic bias for a second kink to form opposite the first kink (F). It should be noted that this process would only be enhanced by negative torsional stress remaining in the minicircle after formation of the first kink.
Figure 6. Principle of cooperative kinking. (A) In a DNA minicircle with high but subcritical curvature, a flexible site is induced. This site could arise due to kinking resulting, e.g. from dissociation of some proteins that constrain negative supercoiling. ( (more ...)
Our interpretation that underwound 100-bp minicircles adopt configurations with two diametrically opposed kinks originated from numerical simulations using Brownian dynamics. However, these simulations are fundamentally a coarse-grained approach that may not adequately reflect structural changes at the level of individual base pairs. Very recently, Mitchell et al.
) applied atomistic simulations to investigate the effect of torsional stress on DNA minicircles that were ~100
bp in size. DNA minicircles that were underwound comparably with MC100 revealed local torsional destabilization in which, two consecutive base stacks switched from right- to left-handed winding (10
). This number of switched stacks corresponds to our estimation taking into account the torsional relaxation induced by changing from right-handed B-DNA to a left-handed structure with ~13
bp per left-handed turn (15
). However, during the simulation time of 50
ns, the DNA minicircles (starting from a perfect circle) were not observed to form kinks. Presumably, the process of kinking requires a longer time than was explored during these simulations. Interestingly, Mitchell et al.
) also simulated DNA minicircles that were more highly underwound, considering minicircles with ΔLk values of −1 and −1.5. These molecules did undergo kinking at two diametrically opposed sites during the simulated time of 50
ns, which is consistent with our model for structural cooperativity. Though the torsional energy in these minicircles was clearly much higher than the minicircles we have observed by cryo-EM, we consider it possible that the additional torsional energy simply allowed the structural cooperativity to occur in timescales capable of being simulated using atomistic simulations. In this case, we view this result as consistent with our model. Measurements of ellipticity of configurations obtained in these atomistic simulations (coordinates provided by J. Mitchell and S. Harris) revealed that molecules with two kinks have the ellipticity of about 1.3, which is remarkably close to that of 100-bp minicircles as determined by cryo-EM and as simulated by us using Brownian dynamics simulations. We therefore, interpret the minicircle structures modeled by Harris et al.
as strong support for our contention that underwinding destabilized DNA is highly flexible for bending, and that small DNA loops of about 100-bp kink in cooperative manner, where the first kink triggers the second one occurring at opposite site across the small DNA loop.
Whereas, the physical properties of DNA kinking have been the focus of this study, one can only appreciate the significance of our findings by considering the broader biological framework of the problem. We believe that DNA kinking may play an important role in gene regulation. Small DNA loops that are ~100
bp in size are common regulatory elements, formed by loop-forming repressor proteins (49
). In these systems, kinked DNA could serve either direct or indirect roles in repressing transcription by RNA polymerase. Kinks may serve direct roles in gene regulation by physically interfering with the processive synthesis of RNA by a transcribing RNA polymerase. The DNA minicircles observed in this study (including the kinked, MC100) were previously used to demonstrate that transcription elongation by T7 RNA polymerase was repressed by the mechanics of tightly looped DNA alone (26
), though it remains unclear to what degree (if any) kinked DNA played a role in the observations. DNA kinks may also serve an indirect role in regulating transcription. For example, DNA kinks may play a critical role in the binding of loop-forming repressors by making DNA more flexible to bending and thereby reducing the energetic cost of loop formation. In the case of the lactose repressor (LacI), it is known that the DNA duplex is destabilized in a region near the apex of the 93-bp loop that is formed in the absence of other DNA-binding proteins (12
In this regard, LacI offers an illustrative example of a model system where stress-induced DNA structures are known to appear. In the study by Becker et al.
), LacI was challenged in vivo
to form small DNA loops ranging from 63
bp to 91
bp in size. The authors investigated looping by LacI both in the presence and absence of the bacterial histone-like protein HU, which is known to preferentially bind to DNA that is both highly bent (52
) and kinked (53
). Interestingly, loops formed by LacI were stabilized in the presence of HU (51
). The authors also note that DNA looping for the most stable loops does not depend on the size of the loop, contrary to expectations for elastically deforming DNA. It therefore, remains possible that HU simply stabilizes kinks naturally appearing at the apexes of the DNA loops already formed by LacI (12
). The possibility that LacI first forms a kinked DNA loop, which is then followed by HU binding, is supported by the observed independence of looping on loop size. That is, if kinks confer significant flexibility to the DNA template, it would be expected that loop formation should be less dependent on the size of the DNA loop. Remarkably, Becker et al.
also demonstrate that the eukaryotic high mobility group protein (HMG) is capable of partially stabilizing the bacterial loops formed by LacI in Escherichia coli
, despite the fact that HMG is not endogenous to bacteria. Like HU, HMG proteins have been shown to bind preferentially to distorted DNA structures (54
), including highly bent DNA (55
). The study by Becker et al.
supports the possibility that DNA kinks are exploited in the LacI system, both to enhance loop formation by conferring enhanced flexibility and to serve as a specific binding site for DNA-binding proteins that recognize stress-induced DNA structures.
In the context of our cryo-EM study of DNA minicircles, we suggest that the kinks that are known to form by LacI are fundamentally similar in nature to the stress-induced kinks we have observed. We, therefore, propose that kinks sustained within the smallest loops formed by LacI significantly increase the flexibility of DNA, approaching the flexibility of ssDNA. Additionally, since our study addresses fundamental, mechanical properties of DNA, the observation of destabilized structures forming in DNA that is highly bent and underwound raises the possibility that DNA kinking serves an important role in many other systems where DNA-binding proteins are known to significantly distort DNA.