Knotting dynamics during DNA replication

The topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two-dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.     

A model of extravascular bubble evolution: effect of changes in breathing gas composition.

Observations of bubble evolution in rats after decompression from air dives (O. Hyldegaard and J. Madsen. Undersea Biomed. Res. 16: 185-193, 1989; O. Hyldegaard and J. Madsen. Undersea Hyperbaric Med. 21: 413-424, 1994; O. Hyldegaard, M. Moller, and J. Madsen. Undersea Biomed. Res. 18: 361-371, 1991) suggest that bubbles may resolve more safely when the breathing gas is a heliox mixture than when it is pure O(2). This is due to a transient period of bubble growth seen during switches to O(2) breathing. In an attempt to understand these experimental results, we have developed a multigas-multipressure mathematical model of bubble evolution, which consists of a bubble in a well-stirred liquid. The liquid exchanges gas with the bubble via diffusion, and the exchange between liquid and blood is described by a single-exponential time constant for each inert gas. The model indicates that bubbles resolve most rapidly in spinal tissue, in adipose tissue, and in aqueous tissues when the breathing gas is switched to O(2) after surfacing. In addition, the model suggests that switching to heliox breathing may prolong the existence of the bubble relative to breathing air for bubbles in spinal and adipose tissues. Some possible explanations for the discrepancy between model and experiment are discussed.     

Differential reaction kinetics, cleavage complex formation, and nonamer binding domain dependence dictate the structure-specific and sequence-specific nuclease activity of RAGs

During V(D)J recombination, RAG (recombination-activating gene) complex cleaves DNA based on sequence specificity. Besides its physiological function, RAG has been shown to act as a structure-specific nuclease. Recently, we showed that the presence of cytosine within the single-stranded region of heteroduplex DNA is important when RAGs cleave on DNA structures. In the present study, we report that heteroduplex DNA containing a bubble region can be cleaved efficiently when present along with a recombination signal sequence (RSS) in cis or trans configuration. The sequence of the bubble region influences RAG cleavage at RSS when present in cis. We also find that the kinetics of RAG cleavage differs between RSS and bubble, wherein RSS cleavage reaches maximum efficiency faster than bubble cleavage. In addition, unlike RSS, RAG cleavage at bubbles does not lead to cleavage complex formation. Finally, we show that the "nonamer binding region," which regulates RAG cleavage on RSS, is not important during RAG activity in non-B DNA structures. Therefore, in the current study, we identify the possible mechanism by which RAG cleavage is regulated when it acts as a structure-specific nuclease.     

Effect of oxygen breathing on micro oxygen bubbles in nitrogen-depleted rat adipose tissue at sea level and 25 kPa altitude exposures

The standard treatment of altitude decompression sickness (aDCS) caused by nitrogen bubble formation is oxygen breathing and recompression. However, micro air bubbles (containing 79% nitrogen), injected into adipose tissue, grow and stabilize at 25 kPa regardless of continued oxygen breathing and the tissue nitrogen pressure. To quantify the contribution of oxygen to bubble growth at altitude, micro oxygen bubbles (containing 0% nitrogen) were injected into the adipose tissue of rats depleted from nitrogen by means of preoxygenation (fraction of inspired oxygen = 1.0; 100%) and the bubbles studied at 101.3 kPa (sea level) or at 25 kPa altitude exposures during continued oxygen breathing. In keeping with previous observations and bubble kinetic models, we hypothesize that oxygen breathing may contribute to oxygen bubble growth at altitude. Anesthetized rats were exposed to 3 h of oxygen prebreathing at 101.3 kPa (sea level). Micro oxygen bubbles of 500-800 nl were then injected into the exposed abdominal adipose tissue. The oxygen bubbles were studied for up to 3.5 h during continued oxygen breathing at either 101.3 or 25 kPa ambient pressures. At 101.3 kPa, all bubbles shrank consistently until they disappeared from view at a net disappearance rate (0.02 mm(2) × min(-1)) significantly faster than for similar bubbles at 25 kPa altitude (0.01 mm(2) × min(-1)). At 25 kPa, most bubbles initially grew for 2-40 min, after which they shrank and disappeared. Four bubbles did not disappear while at 25 kPa. The results support bubble kinetic models based on Fick's first law of diffusion, Boyles law, and the oxygen window effect, predicting that oxygen contributes more to bubble volume and growth during hypobaric conditions. As the effect of oxygen increases, the lower the ambient pressure. The results indicate that recompression is instrumental in the treatment of aDCS.     

A model for influence of exercise on formation and growth of tissue bubbles during altitude decompression

In response to exercise performed before or after altitude decompression, physiological changes are suspected to affect the formation and growth of decompression bubbles. We hypothesized that the work to change the size of a bubble is done by gas pressure gradients in a macro- and microsystem of thermodynamic forces and that the number of bubbles formed through time follows a Poisson process. We modeled the influence of tissue O(2) consumption on bubble dynamics in the O(2) transport system in series against resistances, from the alveolus to the microsystem containing the bubble and its surrounding tissue shell. Realistic simulations of experimental decompression procedures typical of actual extravehicular activities were obtained. Results suggest that exercise-induced elevation of O(2) consumption at altitude leads to bubble persistence in tissues. At the same time, exercise-enhanced perfusion leads to an overall suppression of bubble growth. The total volume of bubbles would be reduced unless increased tissue motion simultaneously raises the rate of bubble formation through cavitation processes, thus maintaining or increasing total bubble volume, despite the exercise.     

The flexibility of locally melted DNA

Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23°C to 42°C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. However, these bubbles are not flexible enough to explain the recently observed very sharp bends in DNA.     

Theoretical study of sequence-dependent nanopore unzipping of DNA

We theoretically investigate the unzipping of DNA electrically driven through a nanometer-size pore. Taking the DNA base sequence explicitly into account, the unpairing and translocation process is described by a biased random walk in a one-dimensional energy landscape determined by the sequential basepair opening. Distributions of translocation times are numerically calculated as a function of applied voltage and temperature. We show that varying these two parameters changes the dynamics from a predominantly diffusive behavior to a dynamics governed by jumps over local energy barriers. The work suggests experimentally studying sequence effects, by comparing the average value and standard deviation of the statistical distribution of translocation times.     

Effect of combined recompression and air, oxygen, or heliox breathing on air bubbles in rat tissues.

The fate of bubbles formed in tissues during the ascent from a real or simulated air dive and subjected to therapeutic recompression has only been indirectly inferred from theoretical modeling and clinical observations. We visually followed the resolution of micro air bubbles injected into adipose tissue, spinal white matter, muscle, and tendon of anesthetized rats recompressed to and held at 284 kPa while rats breathed air, oxygen, heliox 80:20, or heliox 50:50. The rats underwent a prolonged hyperbaric air exposure before bubble injection and recompression. In all tissues, bubbles disappeared faster during breathing of oxygen or heliox mixtures than during air breathing. In some of the experiments, oxygen breathing caused a transient growth of the bubbles. In spinal white matter, heliox 50:50 or oxygen breathing resulted in significantly faster bubble resolution than did heliox 80:20 breathing. In conclusion, air bubbles in lipid and aqueous tissues shrink and disappear faster during recompression during breathing of heliox mixtures or oxygen compared with air breathing. The clinical implication of these findings might be that heliox 50:50 is the mixture of choice for the treatment of decompression sickness.     

Sequence-dependent cost for Z-form shapes the torsion-driven B–Z transition via close interplay of Z-DNA and DNA bubble

Despite recent genome-wide investigations of functional DNA elements, the mechanistic details about their actions remain elusive. One intriguing possibility is that DNA sequences with special patterns play biological roles, adopting non-B-DNA conformations. Here we investigated dynamics of thymine-guanine (TG) repeats, microsatellite sequences and recurrently found in promoters, as well as cytosine–guanine (CG) repeats, best-known Z-DNA forming sequence, in the aspect of Z-DNA formation. We measured the energy barriers of the B–Z transition with those repeats and discovered the sequence-dependent penalty for Z-DNA generates distinctive thermodynamic and kinetic features in the torque-induced transition. Due to the higher torsional stress required for Z-form in TG repeats, a bubble could be induced more easily, suppressing Z-DNA induction, but facilitate the B–Z interconversion kinetically at the transition midpoint. Thus, the Z-form by TG repeats has advantages as a torsion buffer and bubble selector while the Z-form by CG repeats likely behaves as torsion absorber. Our statistical physics model supports quantitatively the populations of Z-DNA and reveals the pivotal roles of bubbles in state dynamics. All taken together, a quantitative picture for the transition was deduced within the close interplay among bubbles, plectonemes and Z-DNA.     

The structure-specific nicking of small heteroduplexes by the RAG complex: implications for lymphoid chromosomal translocations

During V(D)J recombination, the RAG complex binds at recombination signal sequences and creates double-strand breaks. In addition to this sequence-specific recognition of the RSS, the RAG complex has been shown to be a structure-specific nuclease, cleaving 3' overhangs and 3' flaps, and, more recently, 10 nucleotides (nt) bubble (heteroduplex) structures. Here, we assess the smallest size heteroduplex that core and full-length RAGs can cleave. We also test whether bubbles adjacent to a partial RSS are nicked any differently or any more efficiently than bubbles that are surrounded by random sequence. These points are important in considering what types and what size of non-B DNA structure that the RAG complex can nick, and this helps assess the role of the RAG complex in mediating lymphoid chromosomal translocations. We find that the smallest bubble nicked by the RAG complex is 3nt, and proximity to a partial or full RSS sequence does not affect the nicking by RAGs. RAG nicking efficiency increases with the size of the heteroduplex and is only about two-fold less efficient than an RSS when the bubble is 6nt. We consider these findings in the context of RAG nicking at non-B DNA structures in lymphoid chromosomal translocations.     

Analysis of human flap endonuclease 1 mutants reveals a mechanism to prevent triplet repeat expansion

Flap endonuclease 1 (FEN1), involved in the joining of Okazaki fragments, has been proposed to restrain DNA repeat sequence expansion, a process associated with aging and disease. Here we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity. Introduction of these mutants into yeast led to sequence expansions. Reconstituting triplet repeat expansion in vitro, we previously found that DNA ligase I promotes expansion, but FEN1 prevents the ligation that forms expanded products. Here we show that among the intermediates that could generate sequence expansion, a bubble is necessary for ligation to produce the expansion product. Severe exonuclease defects in the mutant FEN1 suggested that the inability to degrade bubbles exonucleolytically leads to expansion. However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated. Instead, we propose that FEN1 suppresses sequence expansion by degrading flaps that equilibrate with bubbles, thereby reducing bubble concentration. In this way FEN1 employs endonuclease rather than exonuclease to prevent expansions. A model is presented describing the roles of DNA structure, DNA ligase I, and FEN1 in sequence expansion.