Dynamics of Small Loops in DNA Molecules

Decision letter: Single-molecule tracking of the transcription cycle by sub-second RNA detection

Single Molecule Studies of Sequence Dependence Elasticity in DNA

By means of atomic force microscopy (AFM), we performed the direct imaging of DNA molecules (200, 500, 1000 bp) in a Tris-borate buffer solution, and measured the contour length and the end-to-end distance of DNA. Processing the data according to the worm-like chain model, we calculated the persistence length of the double-stranded DNA. Based on the analysis of the contour length and the persistence length, we discussed the interactions between DNA and an intercalating fluorescence dye (YO-PRO-1). YO-PRO-1 stacks between the base pairs and extends the contour length of DNA, changing the electric charge and the persistence length of DNA. From AFM measurement, we investigated directly the relationship between the persistence length and the number of the YO-PRO-1 intercalating to DNA. We will discuss on the relationship between the effect of an intercalating dye on the electrophoretic behavior and the conformational changes of DNA with an intercalating dye.

Second-End Capture in DNA Double-Strand Break Repair Promoted by Brh2 Protein of Ustilago maydis

When Computer Simulation Excels Experiment

Genome mapping involves the confinement of long DNA molecules, in excess of 150 kilobase pairs, in nanochannels near the circa 50 nm persistence length of DNA. The fidelity of the map relies on the assumption that the DNA is linearized by channel confinement, which assumes the absence of knots. We have computed the probability of forming different knot types and the size of these knots for long chains (approximately 164 kilobase pairs) via pruned-enriched Rosenbluth method simulations of a discrete wormlike chain model of DNA in channel sizes ranging from 35 nm to 60 nm. Compared to prior simulations of short DNA in similar confinement, these long molecules exhibit both complex knots, with up to seven crossings, and multiple knots per chain. The knotting probability is a very strong function of channel size, ranging from 0.3% to 60%, and rationalized in the context of Odijk's theory for confined semiflexible chains. Overall, the knotting probability and knot size obtained from these equilibrium measurements are not consistent with experimental measurements of the properties of anomalously bright regions along the DNA backbone during genome mapping experiments. This result suggests that these events in experiments are either knots formed during the processing of the DNA prior to injection into the nanochannel or regions of locally high DNA concentration without a topological constraint. If so, knots during genome mapping are not an intrinsic problem for genome mapping technology.

Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58–156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes.

Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50-60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.

Fluorescence nanoscopy of single DNA molecules by using stimulated emission depletion (STED).

In spite of extensive research, the mechanism by which MutS initiates DNA mismatch repair (MMR) remains controversial. We use atomic force microscopy (AFM) to capture how MutS orchestrates the first step of E. coli MMR. AFM images captured two types of MutS/DNA complexes: single-site binding and loop binding. In most of the DNA loops imaged, two closely associated MutS dimers formed a tetrameric complex in which one of the MutS dimers was located at or near the mismatch. Surprisingly, in the presence of ATP, one MutS dimer remained at or near the mismatch site and the other, while maintaining contact with the first dimer, relocated on the DNA by reeling in DNA, thereby producing expanding DNA loops. Our results indicate that MutS tetramers composed of two non-equivalent MutS dimers drive E. coli MMR, and these new observations now reconcile the apparent contradictions of previous 'sliding' and 'bending/looping' models of interaction between mismatch and strand signal.

In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

Internal motion of supercoiled DNA: brownian dynamics simulations of site juxtaposition

Mechanical Constraints on Hin Subunit Rotation Imposed by the Fis/Enhancer System and DNA Supercoiling during Site-Specific Recombination

We have performed canonical ensemble Monte Carlo simulations of a primitive DNA model to study the conformation of 2.56 ~ 21.8 μm long DNA molecules confined in nanochannels at various ionic concentrations with the comparison of our previous experimental findings. In the model, the DNA molecule is represented as a chain of charged hard spheres connected by fixed bond length and the nanochannels as planar hard walls. System potentials consist of explicit electrostatic potential along with short-ranged hard-sphere and angle potentials. Our primitive model system provides valuable insight into the DNA conformation, which cannot be easily obtained from experiments or theories. First, the visualization and statistical analysis of DNA molecules in various channel dimensions and ionic strengths verified the formation of locally coiled structures such as backfolding or hairpin and their significance even in highly stretched states. Although the folding events mostly occur within the region of ~0.5 μm from both chain ends, significant portion of the events still take place in the middle region. Second, our study also showed that two controlling factors such as channel dimension and ionic strength widely used in stretching DNA molecules have different influence on the local DNA structure. Ionic strength changes local correlation between neighboring monomers by controlling the strength of electrostatic interaction (and thus the persistence length of DNA), which leads to more coiled local conformation. On the other hand, channel dimension controls the overall stretch by applying the geometric constraint to the non-local DNA conformation instead of directly affecting local correlation. Third, the molecular weight dependence of DNA stretch was observed especially in low stretch regime, which is mainly due to the fact that low stretch modes observed in short DNA molecules are not readily accessible to much longer DNA molecules, resulting in the increase in the stretch of longer DNA molecules.

Free-radical-induced chain scission and cross-linking of polymers in aqueous solution—an overview