Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

We use cryo-electron microscopy (cryo-EM) to study the 3D shapes of 94-bp-long DNA minicircles and address the question of whether cyclization of such short DNA molecules necessitates the formation of sharp, localized kinks in DNA or whether the necessary bending can be redistributed and accomplished within the limits of the elastic, standard model of DNA flexibility. By comparing the shapes of covalently closed, nicked and gapped DNA minicircles, we conclude that 94-bp-long covalently closed and nicked DNA minicircles do not show sharp kinks while gapped DNA molecules, containing very flexible single-stranded regions, do show sharp kinks. We corroborate the results of cryo-EM studies by using Bal31 nuclease to probe for the existence of kinks in 94-bp-long minicircles.     

Homologous pairing in duplex DNA regions and the formation of four-stranded paranemic joints promoted by RecA protein. Effects of gap length and negative superhelicity

RecA protein catalyzes homologous pairing of partially single-stranded duplex DNA and fully duplex DNA to form stable joint molecules. We constructed circular duplex DNA with various defined gap lengths and studied the pairing reaction between the gapped substrate with fully double-stranded DNA. The reaction required a stoichiometric amount of RecA protein, and the optimal reaction was achieved at a ratio of 1 RecA monomer per 4 base pairs. The length of the gap, ranging from 141 to 1158 nucleotides, had little effect on the efficiency of homologous pairing. By using a circular gapped duplex DNA prepared from the chimeric phage M13Gori1, we were able to show the formation of nonintertwined or paranemic joints in duplex regions between the gapped and fully duplex molecules. The formation of such paranemic joints occurred efficiently and included nearly all of the DNA in the reaction mixture. The reaction required negative superhelicity, and pairing was greatly reduced with linear or nicked circular DNA. We conclude that one functional role of the single-stranded gap is for facilitating the binding of RecA protein to the duplex region of the gapped DNA. Once the nucleoprotein filament is formed, homologous pairing between the gapped and fully duplex DNA can take place anywhere along the length of the nucleoprotein complex.     

Multimerization-cyclization of DNA fragments as a method of conformational analysis

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.     

Ring closure probabilities for DNA fragments by Monte Carlo simulation

The rate of ligation of DNA molecules into circular forms depends on the ring closure probability, commonly called the j-factor, which is a sensitive measure of the extent to which thermal fluctuations contribute to bending and twisting of DNA molecules in solution. We present a theoretical treatment of the cyclization equilibria of DNA that employs a special Monte Carlo method for generating large ensembles of model DNA chains. Using this method, the chain length dependence of the j-factor was calculated for molecules. in the size range 250 to 2000 base-pairs. The Monte Carlo results are compared with recent analytical theory and experimental data. We show that a value of 475 A for the persistence length of DNA, close to values measured by a number of other methods, is in excellent agreement with the cyclization results. Preliminary applications of the Monte Carlo method to the problem of systematically bent DNA molecules are presented. The calculated j-factor is shown to be very sensitive to the amount of bending in these fragments. This fact suggests that ligase closure measurements of systematically bent DNA molecules should be a useful method for studying sequence-directed bending in DNA.     

RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.     

Dynamic bending rigidity of DNA

Rapidly relaxing components in the decay of the transient electric dichroism of DNA restriction fragments were reported by Diekmann et al. [(1982) Biophys. Chem. 15, 263-270] and P?rschke et al. [(1987) Biopolymers 26, 1971-1974]. These are analyzed using a new normal mode theory for weakly bending rods and assigned to bending. The longest bending relaxation times for fragments with 95-250 base pairs coincide with the theoretical curve calculated for a dynamic bending rigidity corresponding to a dynamic persistence length Pd = 2100 A. Analysis of the relative amplitudes of fast and slow components following weak orienting pulses is also consistent with a rather large dynamic persistence length. The enhancement of the relative amplitude of the fast component in large electric fields is attributed to steady-state bending of initially perpendicular DNAs by the field. Several reasons are proposed why the dynamic bending rigidity is 4 times larger than the apparent static bending rigidity inferred from equilibrium persistence length measurements on the same fragments.     

Sequence-dependent elastic properties of DNA

Harmonic elastic constants of 3-11 bp duplex DNA fragments were evaluated using four 5 ns unrestrained molecular dynamics simulation trajectories of 17 bp duplexes with explicit inclusion of solvent and counterions. All simulations were carried out with the Cornell et al. force-field and particle mesh Ewald method for long-range electrostatic interactions. The elastic constants including anisotropic bending and all coupling terms were derived by analyzing the correlations of fluctuations of structural properties along the trajectories. The following sequences have been considered: homopolymer d(ApA)(n) and d(GpG)(n), and alternating d(GPC)(n) and d(APT)(n). The calculated values of elastic constants are in very good overall agreement with experimental values for random sequences. The atomic-resolution molecular dynamics approach, however, reveals a pronounced sequence-dependence of the stretching and torsional rigidity of DNA, while sequence-dependence of the bending rigidity is smaller for the sequences considered. The earlier predicted twist-bend coupling emerged as the most important cross-term for fragments shorter than one helical turn. The calculated hydrodynamic relaxation times suggest that damping of bending motions may play a role in molecular dynamics simulations of long DNA fragments. A comparison of elasticity calculations using global and local helicoidal analyses is reported. The calculations reveal the importance of the fragment length definition. The present work shows that large-scale molecular dynamics simulations represent a unique source of data to study various aspects of DNA elasticity including its sequence-dependence.     

The application of ultrasound as a rapid method to provide DNA fragments suitable for detection by DNA biosensors

Contamination of food and water supplies by microorganisms such as Escherichia coli, the need for point-of-care bedside analysis of biological samples, and concerns about terrorist attacks using biological organisms, have made the development of fast, reliable, and sensitive analytical methodologies for use in monitoring of pathogens very important. With a variety of biosensors being developed for extremely sensitive and rapid nucleic acid diagnostics, it has become even more important to shift focus towards creation of methods to decrease the amount of time and effort necessary for sample preparation. The application of ultrasound has the potential to create DNA fragments from genomic material with lengths that are suitable for determination using biosensors and microarrays. For example, application of 85 W power at a frequency of 20 kHz can produce a preponderance of fragments of 100-400 base pairs (bp) within several seconds, and sample processing can lead to over 75% conversion from genomic material to fragments in times of 20-30 s. A proportion of these fragments are in a single-stranded state and are suitable for hydridization with immobilized single-stranded DNA probe oligonucleotides using a fiber optic biosensor. Control of factors such as salt concentration, exposure time, ultrasound power, and the initial temperature of the solution, can affect the length and form (single- or double-stranded) of DNA fragments that are generated by ultrasound, and average fragment length can be adjusted by selection of these operating parameters.     

Survival of M13mp18 gapped duplex DNA as a function of gap length

Gaps of various lengths were generated in duplex M13mp18DNA by exonuclease III digestion of nicked DNA. The length of the gap increased essentially linearly with time of digestion. Survival in E. coli, however, was not a linear function of gap length. Similar results were obtained when gaps were produced by stopping the polymerization reaction. The survival (N/No) of the gapped DNA in SOS-induced E. coli cells transformed by electroporation and uninduced cells transformed by the calcium chloride method can be quantitatively accounted for by a kinetic model assuming a single-strand endonucleolytic activity (Pd) in the cell which increases linearly with gap length (L) and a repair activity by a polymerase (Pr) which is independent of gap length (formula 1). With uninduced cells transformed by electroporation the results can be mathematically described if assumptions are made concerning the protection of single-stranded parts of the DNA by single-strand affinic proteins.     

Translesion synthesis past platinum DNA adducts by human DNA polymerase mu

DNA polymerase mu (pol mu) is a member of the pol X family of DNA polymerases, and it shares a number of characteristics of both DNA polymerase beta (pol beta) and terminal deoxynucleotidyl transferase (TdT). Because pol beta has been shown to perform translesion DNA synthesis past cisplatin (CP)- and oxaliplatin (OX)-GG adducts, we determined the ability of pol mu to bypass these lesions. Pol mu bypassed CP and OX adducts with an efficiency of 14-35% compared to chain elongation on undamaged DNA, which is second only to pol eta in terms of bypass efficiency. The relative ability of pol mu to bypass CP and OX adducts was dependent on both template structure and sequence context. Since pol mu has been shown to be more efficient on gapped DNA templates than on primed single-stranded DNA templates, we determined the ability of pol mu to bypass Pt-DNA adducts on both primed single-stranded and gapped templates. The bypass of Pt-DNA adducts by pol mu was highly error-prone on all templates, resulting in 2, 3, and 4 nt deletions. We postulate that bypass of Pt-DNA adducts by pol mu may involve looping out the Pt-GG adduct to allow chain elongation downstream of the adduct. This reaction appears to be facilitated by the presence of a downstream "acceptor" and a gap large enough to provide undamaged template DNA for elongation past the adduct, although gapped DNA is clearly not required for bypass.     

Relating independent measures of DNA curvature: electrophoretic anomaly and cyclization efficiency

Electrophoretic methods are often used to measure DNA curvature and protein-induced DNA bending. Though convenient and widely-applied, quantitative analyses are generally limited to assays for which empirical calibration standards have been developed. Alternatively, solution-based cyclization of short DNA duplexes allows analysis of DNA curvature and bending from first principles, but a detailed understanding of this assay is still lacking. In this work, we demonstrate that calibration with an independent electrophoretic assay of DNA curvature permits interpretation of cyclization assay results in a quantitatively meaningful way. We systematically measure intrinsic DNA curvature in short duplexes using a well-established empirical ligation ladder assay. We then compare the results to those obtained from the analysis of the distribution of circular products obtained in simple enzymatic cyclization assays of the same duplexes when polymerized. A strong correlation between DNA curvature estimates from these two assays is obtained for DNA fragments between 150-300 bp in length. We discuss how this result might be used to improve quantitative analysis of protein-mediated bending events evaluated by cyclization methods. Our results suggest that measurements of DNA curvature obtained under similar conditions, in solution and in an acrylamide gel matrix, can be compared directly. The ability to correlate results of these simple assays may prove convenient in monitoring DNA curvature and flexibility.     

Self-assembly of DNA on a gapped carbon nanotube

We perform molecular dynamics simulations to analyze the wrapping process of a single-stranded (ss) DNA around a gapped CNT immersed in a bath of water. We observe the formation of a stable molecular junction with the ssDNA adopting a helical or circular conformation around one CNT electrode and a linear conformation around the opposite electrode. We find that DNA undergoes several conformational changes during equilibration of the self-assembled molecular junction. This process would allow a higher yield of successful CNT-DNA interconnections, which constitutes a novel structure of interest in chemical and biological sensing at the single-molecule level.     

Underwinding of DNA associated with duplex-duplex pairing by RecA protein

Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.     

RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA.

In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end. Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.     

Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro.

The gapped duplex DNA approach to oligonucleotide-directed construction of mutations (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) has been developed further. A procedure is described that makes in vitro DNA polymerase/DNA ligase reactions dispensable. Direct transfection of host bacteria with gdDNA molecules of recombinant phage M13 plus mutagenic oligonucleotide results in marker yields in excess of 50% (gap size 1640 nucleotides). An important feature incorporated into the mutagenic oligonucleotide is the presence of one or two internucleotidic phosphorothioate linkages immediately adjacent to the 5'-terminus. Automated preparation and biochemical properties of such compounds are described as well as their performance in oligonucleotide-directed mutagenesis. A systematic study of the following parameters influencing marker yield is reported: Gap size, length of oligonucleotide, chemical nature of oligonucleotide termini and heatshock temperature during transformation.     

Energetics and specificity of Rat DNA polymerase beta interactions with template-primer and gapped DNA substrates

Interactions between rat polymerase beta (pol beta) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol beta complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNA-binding modes of the enzyme, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotides occluded by the protein. The 8-kDa domain of the enzyme engages the double-stranded (ds)DNA downstream from the primer, while the 31-kDa domain has similar affinity for the ss-ds DNA junction and the dsDNA. The affinity of rat pol beta for the gapped DNA is not affected by the size of the gap. The results indicate a plausible model for recognition of the gapped DNA by rat pol beta. The enzyme binds the ss-ds DNA junction of the gap using the 31-kDa domain. This binding induces an allosteric transition, resulting in the association of the 8-kDa domain with the dsDNA, leading to an amplification of the affinity for the gap. The 5' terminal phosphate, downstream from the primer, has little effect on the affinity, but affects the ssDNA conformation of the gap.     

The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles).

We have studied the structure and reactivities of two kinds of mismatched DNA sequences--unopposed bases, or bulges, and multiple mismatched pairs of bases. These were generated in a constant sequence environment, in relatively long DNA fragments, using a technique based on heteroduplex formation between sequences cloned into single-stranded M13 phage. The mismatched sequences were studied from two points of view, viz 1. The mobility of the fragments on gel electrophoresis in polyacrylamide was studied in order to examine possible bending of the DNA due to the presence of the mismatch defect. Such bending would constitute a global effect on the conformation of the molecule. 2. Sequences in and around the mismatches were studied using enzyme and chemical probes of DNA structure. This would reveal more local structural effects of the mismatched sequences. We observed that the structures of the bulges and the multiple mismatches appear to be fundamentally different. The bulged sequences exhibited a large gel retardation, consistent with a significant bending of the DNA at the bulge, and whose magnitude depends on the number of mismatched bases. The larger bulges were sensitive to cleavage by single-strand specific nucleases, and modified by diethyl pyrocarbonate (adenines) or osmium tetroxide (thymines) in a non-uniform way, suggesting that the bulges have a precise structure that leads to exposure of some, but not all, of the bases. In contrast the multiple mismatches ('bubbles') cause very much less bending of the DNA fragment in which they occur, and uniform patterns of chemical reactivity along the length of the mismatched sequences, suggesting a less well defined, and possibly flexible, structure. The precise structure of the bulges suggests that such features may be especially significant for recognition by proteins.     

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5'-AACNNNNNNGTGC-3', where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequence-specific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a double-stranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response.