PerspectiveMolecular Biology

Unraveling DNA Condensation with Optical Tweezers

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Science  09 Jul 2004:
Vol. 305, Issue 5681, pp. 188-190
DOI: 10.1126/science.1100603

Since the invention of optical tweezers by Ashkin, Chu, and co-workers almost 20 years ago (1), this technique has been applied to a variety of biological systems. It has yielded a wealth of information, such as how biopolymers behave under stress (2), how DNA-interacting enzymes decode or digest DNA (36), how motor proteins walk along molecular tracks (7, 8), and how RNA and protein molecules fold and unfold (911). By allowing the manipulation of individual molecules, optical tweezers reveal how these biological systems work at the molecular level. On page 222 of this issue, Case et al. (12) demonstrate yet another elegant application of this technique: unraveling the mystery of how proteins called condensins make DNA take on a compact form.

In essence, optical tweezers use a tightly focused laser beam to trap a particle in three dimensions and, through redirection of the beam, manipulate the particles (1). Focused light exerts two forces on the particle. The gradient force draws the particle toward the focus of the beam where the light field is the strongest. The scattering force arises from the radiation pressure exerted on the particle by absorbed or scattered photons, which blow the particle down the optical axis like a wind. When balanced, these two forces hold the particle just slightly downstream of the light focus.

Perhaps the most fascinating applications of optical tweezers are in biology. Because biological molecules are often too small to be manipulated directly by optical tweezers, a micrometer-sized dielectric sphere is often attached to the molecule of interest to serve as a handle (see the figure, panel A). This allows one to manipulate the position of the attached biomolecule and to exert a well-defined force. Both parameters can be determined with great accuracy: The position of the microsphere can be measured to within 1 nm, and the force to within 1 pN. Optical tweezers, therefore, allow exquisite control over the manipulation of biomolecules.

Using this technique, Case et al. have discovered that bacterial condensins compact DNA in an orderly fashion. DNA condensation is essential for cell division because compact DNA is much easier to split between two daughter cells than DNA in its expanded form. The chromosomal DNA of bacteria is compacted into a nucleoid (13), and eukaryotic cells employ an elaborate mitotic machinery to achieve chromosome compaction (14). In both worlds, SMC (Structural Maintenance of Chromosomes) proteins are crucial players in the process of DNA condensation (13, 14).

Case and co-workers concentrated on the Escherichia coli condensin MukBEF, which consists of an SMC dimer (MukB) and two non-SMC subunits (MukE and MukF) (see the figure, panel A). In their setup, polystyrene beads were attached to the two ends of a piece of double-stranded DNA. One bead was held by an optical trap and the other by a micropipette (panel A). By moving the micropipette away from the optical beam, the investigators obtained a force-versus-extension curve for the DNA. In a previous study, this group had used a similar method to examine the mechanical properties of DNA. However, when they applied their trick to DNA in the presence of MukBEF and ATP, they were caught by surprise.

First, they found that the force-extension curve increased more quickly than observed for naked DNA (see the figure, panel B). This is perhaps not so surprising considering that MukBEF condenses DNA. A very interesting phenomenon, however, occurred as the force reached 17 pN: A sawtooth pattern became superimposed on an otherwise flat region of the curve, indicating that the DNA-MukBEF complex underwent a phase transition consisting of individual decondensation events (see the figure, panel B). As the force was relaxed, the DNA returned to its original condensed form. Amazingly, when the applied force was less than but still close to 17 pN, recondensation was so slow that individual condensation steps of 35 nm were observed, probably caused by the conformational change of a single MukBEF molecule. Condensation took place in the presence of ATP and its nonhydrolyzable analogs but not in their absence, indicating that ATP binding rather than ATP hydrolysis provided the energy source for condensation.

A nip and tuck for DNA.

Unraveling a DNA-MukBEF complex with optical tweezers. (A) In the experimental setup of Case et al. (12), polystyrene beads (beige spheres) were attached to the two ends of a piece of double-stranded DNA (purple). One bead was held by an optical trap (orange) and the other by a micropipette (gray handle). By moving the micropipette away from the optical beam, the investigators obtained force-versus-extension curves for DNA bound to the bacterial condensin MukBEF (blue). Here, only the SMC dimers (MukB) are shown; the non-SMC subunits, MukE and MukF, are believed to bind to the head domains of MukB (blue rectangles). (B) Force-versus-extension curves obtained for three consecutive pulling runs of the DNA-MukBEF complex. (C) A model of force-induced decondensation of the DNA-MukBEF complex.

Perhaps the most surprising result is the reproducible sawtooth pattern observed when the same DNA was pulled multiple times. Sawtooth patterns in force-extension curves of biopolymers are not a new phenomenon. When the giant muscle protein, titin, was stretched by an atomic force apparatus, a beautiful sawtooth pattern was observed, indicating the sequential unfolding of individual immunoglobulin domains (15). A similar pattern was also seen when a nucleosome array was pulled by an optical trap, signaling the release of DNA from individual histones (16). In these examples, the sawtooth pattern indicated a trend toward increasing disruption forces. This would be expected given that higher forces are required to break stronger bonds and so such bonds tend to break later during the force loading process. For bonds that require a similar amount of force to break, individual disruptions are believed to occur randomly. However, in the case of the DNA-MukBEF complex, the investigators obtained a highly reproducible sawtooth pattern in consecutive pulling runs with no correlation between the disruption force of each peak and its order of occurrence (see the figure, panel B).

To explain these results, Case and co-workers propose that ATP-bound MukBEF polymerizes along the DNA by interactions between the head domains of adjacent MukBEF molecules (see the figure, panel C). This joint-head structure binds tightly to DNA (panel C) and does not detach from the DNA even under forces as high as 90 pN. This ATP-dependent interaction between the two neighboring head domains of MukBEF and the subsequent stimulation of binding between DNA and the SMC dimers is consistent with a recent biochemical study of the Bacillus subtilis SMC (17). Case et al. further propose that a weaker intramolecular interaction between the two heads of the same MukBEF dimer causes DNA condensation. The breaking of this interaction at 17 pN leads to the observed sawtooth pattern (see the figure, panel C). The striking reproducibility of the sawtooth pattern may indicate that this latter interaction always starts to break from one end of the condensed DNA filament, presumably because the force is weaker for a MukBEF molecule that has only a single neighbor (panel C).

Although these experiments have provided new insights into the interactions between condensins and DNA that are critical for understanding the DNA condensation process, much is still unknown. For example, the compaction ratio observed in this experiment is only about 4, much less than the observed value of 103 to 104 in bacteria. Concerted actions of condensins, topoisomerases, other DNA binding proteins, and DNA supercoiling in vivo are likely to be responsible for this discrepancy, but the interplay between these different factors is largely unknown. Another interesting question is whether the functional mechanism of DNA condensation orchestrated by condensins is conserved between prokaryotic and eukaryotic cells. Considering the largely conserved structure of SMC dimers between the two types of organisms, one would be inclined to say yes. However, a recent single-molecule study of Xenopus laevis condensin I by Strick and co-workers gave very different results (18). The most striking difference is that ATP hydrolysis is required for the compaction of DNA by condensin I, whereas Case et al. show that MukBEF-induced DNA condensation takes place in the presence of nonhydrolyzable ATP as well. Quantitatively, the step sizes of individual condensation and decondensation events are noticeably larger for condensin I, and so is the overall compaction ratio observed in the experiment by Strick and co-workers. Whether these differences arise from different functional mechanisms between prokaryotic and eukaryotic condensins or are merely due to different experimental conditions is worth investigating.

Having observed many elegant experiments on biological molecules using optical tweezers, I've often wondered how far this method can take us. After all, holding a molecule at its two ends and then stretching it is not what happens to biomolecules naturally in cells. However, the power of this technique has never ceased to amaze me when applied with intelligent experimental design. For example, efforts to improve the precision of optical traps now allow the transcription of DNA to be followed at almost base-pair resolution (19). Processes as complicated as DNA packaging by viruses and DNA uptake by bacteria have been studied under near physiological conditions (20, 21). Case et al. have now added another wonderful example to this list, not to mention the equally impressive accomplishments attributable to other single-molecule force spectroscopy techniques (2224). In the field of manipulation and force measurements of single molecules, it appears that, so far, one is limited only by one's imagination.

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