C-DNA may facilitate homologous DNA pairing.

Recombination-independent homologous pairing represents a prominent yet largely enigmatic feature of chromosome biology. As suggested by studies in the fungus Neurospora crassa, this process may be based on the direct pairing of homologous DNA molecules. Theoretical search for the DNA structures consistent with those genetic results has led to an all-atom model in which the B-DNA conformation of the paired double helices is strongly shifted toward C-DNA. Coincidentally, C-DNA also features a very shallow major groove that could permit initial homologous contacts without atom-atom clashes. The hereby conjectured role of C-DNA in homologous pairing should encourage the efforts to discover its biological functions and may also clarify the mechanism of recombination-independent recognition of DNA homology.

Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa.

The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using "meiotic silencing by unpaired DNA" (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)-dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.

Modulation of C-to-T mutation by recombination-independent pairing of closely positioned DNA repeats.

Repeat-induced point mutation is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. Repeat-induced point mutation detects duplications (irrespective of their origin, specific sequence, coding capacity, and genomic positions) by a recombination-independent mechanism that likely matches intact DNA double helices directly, without relying on the annealing of complementary single strands. In the fungus Neurospora crassa, closely positioned repeats can induce mutation of the adjoining nonrepetitive regions. This process is related to heterochromatin assembly and requires the cytosine methyltransferase DIM-2. Using DIM-2-dependent mutation as a readout of homologous pairing, we find that GC-rich repeats produce a much stronger response than AT-rich repeats, independently of their intrinsic propensity to become mutated. We also report that direct repeats trigger much stronger DIM-2-dependent mutation than inverted repeats. These results can be rationalized in the light of a recently proposed model of homologous DNA pairing, in which DNA double helices associate by forming sequence-specific quadruplex-based contacts with a concomitant release of supercoiling. A similar process featuring pairing-induced supercoiling may initiate epigenetic silencing of repetitive DNA in other organisms, including humans.

Partition of Repeat-Induced Point Mutations Reveals Structural Aspects of Homologous DNA-DNA Pairing.

In some fungi, a premeiotic process known as repeat-induced point mutation (RIP) can accurately identify and mutate nearly all gene-sized DNA repeats present in the haploid germline nuclei. Studies in Neurospora crassa have suggested that RIP detects sequence homology directly between intact DNA double helices, without strand separation and without the participation of RecA-like proteins. Those studies used the aggregated number of RIP mutations as a simple quantitative measure of RIP activity. Additional structural information about homologous DNA-DNA pairing during RIP can be extracted by analyzing spatial distributions of RIP mutations converted into profiles of partitioned RIP propensity (PRP). Further analysis shows that PRP is strongly affected by the topological configuration and the relative positioning of the participating DNA segments. Most notably, pairs of closely positioned repeats produce very distinct PRP profiles depending on whether these repeats are present in the direct or the inverted orientation. Such an effect can be attributed to a topology-dependent redistribution of the supercoiling stress created by the predicted limited untwisting of the DNA segments during pairing. This and other results raise a possibility that such pairing-induced fluctuations in DNA supercoiling can modulate the overall structure and properties of repetitive DNA. Such effects can be particularly strong in the context of long tandem-repeat arrays that are typically present in the pericentromeric and centromeric regions of chromosomes in many species of plants, fungi, and animals, including humans.

Homologous Pairing between Long DNA Double Helices.

Molecular recognition between two double stranded (ds) DNA with homologous sequences may not seem compatible with the B-DNA structure because the sequence information is hidden when it is used for joining the two strands. Nevertheless, it has to be invoked to account for various biological data. Using quantum chemistry, molecular mechanics, and hints from recent genetics experiments, I show here that direct recognition between homologous dsDNA is possible through the formation of short quadruplexes due to direct complementary hydrogen bonding of major-groove surfaces in parallel alignment. The constraints imposed by the predicted structures of the recognition units determine the mechanism of complexation between long dsDNA. This mechanism and concomitant predictions agree with the available experimental data and shed light upon the sequence effects and the possible involvement of topoisomerase II in the recognition.

Atomic force microscopy study of DNA flexibility on short length scales: smooth bending versus kinking.

The apparently anomalous flexibility of DNA on short length scales has attracted a lot of attention in recent years. We use atomic force microscopy (AFM) in solution to directly study the DNA bending statistics for small lengths down to one helical turn. The accuracy of experimental estimates could be improved due to a large data volume and a refined algorithm for image processing and measuring bend angles. It is found that, at length scales beyond two helical turns (7 nm), DNA is well described by the harmonic worm-like chain (WLC) model with the bending persistence length of 56 nm. Below this threshold, the AFM data are also described by the WLC model assuming that the accuracy of measured bend angles is limited by the physical width of the double helix. We conclude that the double helical DNA behaves as a uniform elastic rod even at very short length scales. Strong bends due to kinks, melting bubbles and other deviations from the WLC model are statistically negligible.

Algebraic Statistics of Poincaré Recurrences in a DNA Molecule.

The statistics of Poincaré recurrences is studied for the base-pair breathing dynamics of an all-atom DNA molecule in a realistic aqueous environment with thousands of degrees of freedom. It is found that at least over five decades in time the decay of recurrences is described by an algebraic law with the Poincaré exponent close to ß=1.2. This value is directly related to the correlation decay exponent ν=ß-1, which is close to ν≈0.15 observed in the time resolved Stokes shift experiments. By applying the virial theorem we analyze the chaotic dynamics in polynomial potentials and demonstrate analytically that an exponent ß=1.2 is obtained assuming the dominance of dipole-dipole interactions in the relevant DNA dynamics. Molecular dynamics simulations also reveal the presence of strong low frequency noise with the exponent η=1.6. We trace parallels with the chaotic dynamics of symplectic maps with a few degrees of freedom characterized by the Poincaré exponent ß~1.5.

DNA flexibility on short length scales probed by atomic force microscopy.

Unusually high bending flexibility has been recently reported for DNA on short length scales. We use atomic force microscopy (AFM) in solution to obtain a direct estimate of DNA bending statistics for scales down to one helical turn. It appears that DNA behaves as a Gaussian chain and is well described by the wormlike chain model at length scales beyond 3 helical turns (10.5 nm). Below this threshold, the AFM data exhibit growing noise because of experimental limitations. This noise may hide small deviations from the Gaussian behavior, but they can hardly be significant.

Mycoplasma gallisepticum produces a histone-like protein that recognizes base mismatches in DNA.

Mycoplasmas are the smallest known microorganisms, with drastically reduced genome sizes. One of the essential biochemical pathways lost in mycoplasmas is methylation-mediated DNA repair (MMR), which is responsible for correction of base substitutions, insertions, and deletions in both bacteria and higher organisms. We found that the histone-like protein encoded by the himA/hup_2 gene of Mycoplasma gallisepticum (mgHU) recognizes typical MMR substrates, in contrast to homologues from other species. The recognition of substitution mismatches is sequence-dependent, with affinities decreasing in the following order: CC > CT = TT > AA = AC. Insertions or deletions of one nucleotide are also specifically recognized with the following sequence-dependent preference: A = T > C. One-nucleotide lesions involving guanine are bound only weakly, and this binding is indistinguishable from binding to intact DNA. Although mgHU is dissimilar to Escherichia coli HU, expression in a slow-growing hupAB E. coli strain restores wild-type growth. The results indicate that mgHU executes all essential functions of bacterial architectural proteins. The origin and the possible role of enhanced specificity for typical MMR substrates are discussed.

Anharmonic torsional stiffness of DNA revealed under small external torques.

DNA supercoiling plays an important role in a variety of cellular processes. The torsional stress related to supercoiling may also be involved in gene regulation through the local structure and dynamics of the double helix. To check this possibility, steady torsional stress was applied in the course of all-atom molecular dynamics simulations of two DNA fragments with different base pair sequences. For one fragment, the torsional stiffness significantly varied with small twisting. The effect is traced to sequence-specific asymmetry of local torsional fluctuations, and it should be small in long random DNA due to compensation. In contrast, the stiffness of special short sequences can change significantly, which gives a simple possibility of gene regulation via probabilities of strong fluctuations. These results have important implications for the role of DNA twisting in complexes with transcription factors.

HU binds and folds single-stranded DNA.

The nucleoid-associated protein HU plays an important role in bacterial nucleoid organization and is involved in numerous processes including transposition, recombination and DNA repair. We show here that HU binds specifically DNA containing mismatched region longer than 3 bp as well as DNA bulges. HU binds single-stranded DNA (ssDNA) in a binding mode that is reminiscent but different from earlier reported specific HU interactions with double-helical DNA lesions. An HU dimer requires 24 nt of ssDNA for initial binding, and 12 nt of ssDNA for each additional dimer binding. In the presence of equimolar amounts of HU dimer and DNA, the ssDNA molecule forms an U-loop (hairpin-like) around the protein, providing contacts with both sides of the HU body. This mode differs from the binding of the single-strand-binding protein (SSB) to ssDNA: in sharp contrast to SSB, HU binds ssDNA non-cooperatively and does not destabilize double-helical DNA. Furthermore HU has a strong preference for poly(dG), while binding to poly(dA) is the weakest. HU binding to ssDNA is probably important for its capacity to cover and protect bacterial DNA both intact and carrying lesions.

Direct Homologous dsDNA-dsDNA Pairing: How, Where, and Why?

The ability of homologous chromosomes (or selected chromosomal loci) to pair specifically in the apparent absence of DNA breakage and recombination represents a prominent feature of eukaryotic biology. The mechanism of homology recognition at the basis of such recombination-independent pairing has remained elusive. A number of studies have supported the idea that sequence homology can be sensed between intact DNA double helices in vivo. In particular, recent analyses of the two silencing phenomena in fungi, known as "repeat-induced point mutation" (RIP) and "meiotic silencing by unpaired DNA" (MSUD), have provided genetic evidence for the existence of the direct homologous dsDNA-dsDNA pairing. Both RIP and MSUD likely rely on the same search strategy, by which dsDNA segments are matched as arrays of interspersed base-pair triplets. This process is general and very efficient, yet it proceeds normally without the RecA/Rad51/Dmc1 proteins. Further studies of RIP and MSUD may yield surprising insights into the function of DNA in the cell.

Kinetic and thermodynamic DNA elasticity at micro- and mesoscopic scales.

Recent theoretical and experimental studies have suggested that the elastic behavior of the small-length double-helical DNA does not correspond to the simple harmonic model. This article presents a thorough comparison of classical atom-level molecular dynamics (MD) and coarse-grained harmonic approximations. It is shown that the previously predicted duration of MD trajectories necessary for accurate assessment of DNA elasticity was significantly overestimated and that reliable estimates of elastic parameters can be obtained after a few tens of nanoseconds. The all-atom and coarse-grained ensembles were compared head-to-head, including the amplitudes and relaxation rates of internal fluctuations as well as translational diffusion. The computed diffusion rates were found to be similar, with good correspondence to experimental data. The torsional persistence length (PL) in MD agrees reasonably well with experiment, with the relaxation rate of twisting fluctuations corresponding well to the harmonic model. The bending PL also agrees reasonably well with experiment, but the corresponding relaxation rate is much higher than the harmonic approximation. For a tetradecamer DNA, the difference reaches 1 order of magnitude, with the bending dynamics faster than the twisting dynamics, in qualitative contrast to the coarse-grained model. The possible mechanisms of this anomalous behavior are discussed.

Analysis of accordion DNA stretching revealed by the gold cluster ruler.

A promising method for measuring intramolecular distances in solution uses small-angle x-ray scattering interference between gold nanocrystal labels [Mathew-Fenn, Science 322, 446 (2008)]. When applied to double-stranded DNA, it revealed that the DNA length fluctuations are strikingly strong and correlated over at least 80 base pair steps. In other words, the DNA behaves as accordion bellows with distant fragments stretching and shrinking concertedly. This hypothesis, however, disagrees with earlier experimental and computational observations. This Rapid Communication shows that the discrepancy can be rationalized by taking into account the cluster exclusion volume and assuming a moderate long-range repulsion between them. The long-range interaction can originate from an ion exclusion effect and cluster polarization in close proximity to the DNA surface.

Ion dynamics and water percolation effects in DNA polymorphism.

The dynamics of ions and water at the surface of DNA are studied by computer simulations in a wide range of hydrations involving the zone of low-hydration polymorphism in DNA. The long-range mobility of ions exhibits a stepwise increase at three distinct hydration levels. The first of them is close to the midpoint of the water percolation transition as well as the midpoint of the transition between A- and B-DNA forms. It coincides with the onset of the dissociation of ion pairs on the DNA surface probably caused by the increase in the water dielectric permittivity due to the appearance of the spanning hydrogen-bonding network. The other two steps are attributed to the formation of percolating water layers on the surface of DNA accompanied by the progressive escape of ions from the DNA surface. The results agree with earlier experimental data and further corroborate the suggested universal mechanism of the low hydration polymorphism in DNA including intraduplex electrostatic condensation close to the water percolation threshold.

The electrostatic origin of low-hydration polymorphism in DNA.

In recent years, significant progress has been made towards uncovering the physical mechanisms of low-hydration polymorphism in double-helical DNA. The effect appears to be mechanistically similar in different biological systems, and it is due to the ability of water to form spanning H-bonded networks around biomacromolecules via a quasi-two-dimensional percolation transition. In the case of DNA, disintegration of the spanning H-bonded network leads to electrostatic condensation of DNA strands because, below the percolation threshold, water loses its high dielectric permittivity, whereas the concentration of neutralizing counterions becomes high. In this Concept article arguments propose that this simple electrostatic mechanism represents the universal origin of low-hydration polymorphism in DNA.

Statistics of time-limited ensembles of bent DNA conformations.

The paper considers statistical properties of ensembles of chain conformations obtained by short-time Brownian dynamics (BD) of a coarse-grained DNA model in order to find out if the conditions necessary for accurate evaluation of the polymer elasticity are attainable in atom-level molecular dynamics (MD) simulations. To measure the bending persistence length (PL) with a 10% error using data accumulated in a single trajectory of a double helix of 15 base pairs, dynamics should be continued for a few microseconds. However, these estimates should be scaled down by about 2 orders of magnitude because the bending dynamics of short double helices in MD features much smaller relaxation times. As a result, good qualitative agreement with the worm-like chain (WLC) theory is reached in MD after tens of nanoseconds. The presently accessible durations of MD trajectories provide reasonably accurate evaluation of DNA elasticity and allow modeling of its mesoscopic properties. The surprisingly fast bending dynamics of short double helices in MD suggests that the microscopic mechanisms of DNA flexibility differ from a simple harmonic model.

Water clustering and percolation in low hydration DNA shells.

The hydrogen-bonded networks of water at the surface of a model DNA molecule are analyzed. At low hydrations, only small water clusters are attached to the DNA surface, whereas, at high hydrations, it is homogeneously covered by a spanning water network. The spanning water network is formed via a percolation transition at an intermediate hydration number of about 15 water molecules per nucleotide, which is very close to the midpoint of polymorphic transitions between A- and B-forms of the double helix. The percolation transition can occur in both A- and B-DNA hydration shells with nearly identical percolation thresholds. However, the mechanism of the percolation transition in A- and B-DNA is qualitatively different in regard to the roles played by the two opposite grooves of the double helix. Free ions can shift the percolation threshold by preventing some water molecules from hydrogen bond networking. The results corroborate the suggested relationship between water percolation and the low hydration polymorphism in DNA.

Weak nanoscale chaos and anomalous relaxation in DNA.

Anomalous nonexponential relaxation in hydrated biomolecules is commonly attributed to the complexity of the free-energy landscapes, similarly to polymers and glasses. It was found recently that the hydrogen-bond breathing of terminal DNA base pairs exhibits a slow power-law relaxation attributable to weak Hamiltonian chaos, with parameters similar to experimental data. Here, the relationship is studied between this motion and spectroscopic signals measured in DNA with a small molecular photoprobe inserted into the base-pair stack. To this end, the earlier computational approach in combination with an analytical theory is applied to the experimental DNA fragment. It is found that the intensity of breathing dynamics is strongly increased in the internal base pairs that flank the photoprobe, with anomalous relaxation quantitatively close to that in terminal base pairs. A physical mechanism is proposed to explain the coupling between the relaxation of base-pair breathing and the experimental response signal. It is concluded that the algebraic relaxation observed experimentally is very likely a manifestation of weakly chaotic dynamics of hydrogen-bond breathing in the base pairs stacked to the photoprobe and that the weak nanoscale chaos can represent an ubiquitous hidden source of nonexponential relaxation in ultrafast spectroscopy.