Model for a DNA-mediated synaptic complex suggested by crystal packing of gamma delta resolvase subunits.

The packing arrangement of the 12 subunits of intact gamma delta resolvase in the unit cell of a hexagonal crystal form suggests a model for site-specific recombination that involves a DNA-mediated synaptic intermediate. The crystal structure has been determined by molecular replacement and partially refined at 2.8/3.5 A resolution. Although the small DNA-binding domain is disordered in these crystals, packing considerations show that only a small region of space in the crystal could accommodate a domain of its size. A family of related models for a synaptic complex between two DNA duplexes and 12 monomers that are arranged as situated in the crystal is consistent with the known topology of the complex and the distances between the three resolvase dimer-binding sites per DNA; further, these models place the two DNA recombination sites in contact with each other between two resolvase dimers, implying that strand exchange is accomplished through direct DNA-DNA interaction. A major role postulated, then, for the resolvase protein assembly is to stabilize a res DNA structure that is close to the topological transition state of the reaction.     

Detecting recombination in 4-taxa DNA sequence alignments with Bayesian hidden Markov models and Markov chain Monte Carlo

This article presents a statistical method for detecting recombination in DNA sequence alignments, which is based on combining two probabilistic graphical models: (1) a taxon graph (phylogenetic tree) representing the relationship between the taxa, and (2) a site graph (hidden Markov model) representing interactions between different sites in the DNA sequence alignments. We adopt a Bayesian approach and sample the parameters of the model from the posterior distribution with Markov chain Monte Carlo, using a Metropolis-Hastings and Gibbs-within-Gibbs scheme. The proposed method is tested on various synthetic and real-world DNA sequence alignments, and we compare its performance with the established detection methods RECPARS, PLATO, and TOPAL, as well as with two alternative parameter estimation schemes.     

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.     

Biologically important conformational features of DNA as interpreted by quantum mechanics and molecular mechanics computations of its simple fragments

Deciphering the mechanism of functioning of DNA as the carrier of genetic information requires identifying inherent factors determining its structure and function. Following this path, our previous DFT studies attributed the origin of unique conformational characteristics of right-handed Watson-Crick duplexes (WCDs) to the conformational profile of deoxydinucleoside monophosphates (dDMPs) serving as the minimal repeating units of DNA strand. According to those findings, the directionality of the sugar-phosphate chain and the characteristic ranges of dihedral angles of energy minima combined with the geometric differences between purines and pyrimidines determine the dependence on base sequence of the three-dimensional (3D) structure of WCDs. This work extends our computational study to complementary deoxydinucleotide-monophosphates (cdDMPs) of non-standard conformation, including those of Z-family, Hoogsteen duplexes, parallel-stranded structures, and duplexes with mispaired bases. For most of these systems, except Z-conformation, computations closely reproduce experimental data within the tolerance of characteristic limits of dihedral parameters for each conformation family. Computation of cdDMPs with Z-conformation reveals that their experimental structures do not correspond to the internal energy minimum. This finding establishes the leading role of external factors in formation of the Z-conformation. Energy minima of cdDMPs of non-Watson-Crick duplexes demonstrate different sequence-dependence features than those known for WCDs. The obtained results provide evidence that the biologically important regularities of 3D structure distinguish WCDs from duplexes having non-Watson-Crick nucleotide pairing.     

A general model for site-specific recombination by the integrase family recombinases.

We present here a general model for integrase family site-specific recombination using the geometric relationships of the cleavable phosphodiester bonds and the disposition of the recombinase monomers (defined by their binding planes) with respect to them. The 'oscillation model' is based largely on the conformations of the recombinase-bound DNA duplexes and their dynamics within Holliday junctions. The duplex substrate or the Holliday junction intermediate is capable of 'oscillating' between two cleavage-competent asymmetric states with respect to corres-ponding chemically inert 'equilibrium positions'. The model accommodates several features of the Flp system and predicts two modes of DNA cleavage during a normal recombination event. It is equally applicable to other systems that mediate recombination across 6, 7 or 8 bp long strand exchange regions (or spacers). The model is consistent with approximately 0-1, 1-2 and 2-3 bp of branch migration during recombination reactions involving 6, 7 and 8 bp spacers, respectively.     

The inherent properties of DNA four-way junctions: comparing the crystal structures of holliday junctions

Holliday junctions are four-stranded DNA complexes that are formed during recombination and related DNA repair events. Much work has focused on the overall structure and properties of four-way junctions in solution, but we are just now beginning to understand these complexes at the atomic level. The crystal structures of two all-DNA Holliday junctions have been determined recently from the sequences d(CCGGGACCGG) and d(CCGGTACCGG). A detailed comparison of the two structures helps to distinguish distortions of the DNA conformation that are inherent to the cross-overs of the junctions in this crystal system from those that are consequences of the mismatched dG.dA base-pair in the d(CCGGGACCGG) structure. This analysis shows that the junction itself perturbs the sequence-dependent conformational features of the B-DNA duplexes and the associated patterns of hydration in the major and minor grooves only minimally. This supports the idea that a DNA four-way junction can be assembled at relatively low energetic cost. Both structures show a concerted rotation of the adjacent duplex arms relative to B-DNA, and this is discussed in terms of the conserved interactions between the duplexes at the junctions and further down the helical arms. The interactions distant from the strand cross-overs of the junction appear to be significant in defining its macroscopic properties, including the angle relating the stacked duplexes across the junction.     

Transitions between B-DNA and Z-DNA: a dilemma

The intramolecular conformational change which occurs in double-stranded (dG-dC) oligonucleotides and polynucleotides in high salt solutions is currently assumed to be a transition between the presently accepted structures for B-DNA and Z-DNA. However, a serious dilemma results from the fact that these two structures are from topologically different families of helical duplexes. Mechanisms for interconversion between the two forms, under typical experimental conditions, are highly improbable from a physical-chemical viewpoint. The alternative possibility is that the structure of one of the forms in the transition is not as assumed. The resulting dilemma is addressed and alternative relationships between right- and left-handed DNA forms are discussed.     

DNA branch nuclease activity of vaccinia A22 resolvase

DNA replication, recombination, and repair can result in formation of diverse branched DNA structures. Many large DNA viruses are known to encode DNA branch nucleases, but several of the expected activities have not previously been found among poxvirus enzymes. Vaccinia encodes an enzyme, A22 resolvase, which is known to be active on four-stranded DNA junctions (Holliday junctions) or Holliday junction-like structures containing three of the four strands. Here we report that A22 resolvase in fact has a much wider substrate specificity than previously appreciated. A22 resolvase cleaves Y-junctions, single-stranded DNA flaps, transitions from double strands to unpaired single strands ("splayed duplexes"), and DNA bulges in vitro. We also report site-directed mutagenesis studies of candidate active site residues. The results identify the likely active site and support a model in which a single active site is responsible for cleavage on Holliday junctions and splayed duplexes. Lastly, we describe possible roles for the A22 resolvase DNA-branch nuclease activity in DNA replication and repair.     

Intermediates in extrachromosomal homologous recombination in Xenopus laevis oocytes: characterization by electron microscopy.

Several molecular mechanisms have been proposed to account for nonconservative homologous recombination. This type of recombination is particularly efficient in Xenopus oocytes when appropriate DNA substrates are injected. To distinguish between possible models, we have investigated recombination intermediates from oocytes by direct observation in the electron microscope. Partially recombined DNA was crosslinked with a psoralen derivative after incubation in oocytes to limit the branch migration that might occur during recovery procedures and alter the structures that were initially present. Branched structures, which we interpret as intermediates, represented approximately 10% of the DNA recovered and were readily analyzed. We did not observe any structures with internal loops predicted by invasion mechanisms. The majority of intermediates had one or two single-stranded branches on product-sized molecules, as predicted for incomplete junctions in the resection-annealing mechanism. Detailed length measurements confirmed the expectations of that model. When recovered DNA was not crosslinked, or when annealed junctions were prepared in vitro, we saw branched structures that indicated the occurrence of extensive branch migration. Comparison with the crosslinked sample confirmed the effectiveness of the crosslinking in preserving structures created in the oocytes. Our results strongly support a resection-annealing mechanism of recombination in oocytes.     

The mechanics of winding and unwinding helices in recombination: torsional stress associated with strand transfer promoted by RecA protein

Homologous recombination usually involves the production of heteroduplex DNA, DNA containing strands contributed from two different duplexes. RecA protein of E. coli can promote the formation of heteroduplex DNA in vitro by the exchange of DNA strands between two helical structures, duplex DNA and a helical recA nucleoprotein filament containing a single strand of DNA. Complete unwinding of the parental duplex and the rewinding of one strand with a new complement requires rotation of the helical structures about one another, or about their respective longitudinal axes. The observations described here demonstrate an association of torsional stress with strand exchange, and suggest that exchange is accomplished principally by concomitant rotation of duplex DNA and the recA nucleoprotein filament, each about its longitudinal axis.     

Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family

Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.     

Resolution of synthetic Holliday structures by an extract of human cells.

Virtually all models for recombination between homologous DNA sequences invoke a branched intermediate known as a Holliday structure. The terminal steps of recombination are postulated to involve a specific cleavage through the four-way junction of a Holliday structure, in a process known as resolution. We have constructed a synthetic Holliday structure in which the position of the junction of the DNA duplexes can branch migrate through approximately 185 bp. Using this structure, we have found that a component of a cytoplasmic extract of Hela cells is capable of cleaving the central junction of the substrate in a manner consistent with resolution. The activity requires a divalent cation but does not require an exogenous energy source. This is the first reported resolution activity from a mammalian source.     

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.     

DNA topology and geometry in Flp and Cre recombination

The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the lambda-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13bp inverted repeats flanking a 6 or 8bp spacer sequence. Recombination reactions involve particular geometric and topological relationships between DNA target sites at synapsis, which we investigate using nicked-circular DNA molecules. Examination of the tertiary structure of synaptic complexes formed on nicked plasmid DNAs by atomic-force microscopy, in conjunction with detailed topological analysis using the mathematics of tangles, shows that only a limited number of recombination-site topologies are consistent with the global structures of plasmids bearing directly and inversely repeated sites. The tangle solutions imply that there is significant distortion of the Holliday-junction intermediate relative to the planar structure of the four-way DNA junction present in the Flp and Cre co-crystal structures. Based on simulations of nucleoprotein structures that connect the two-dimensional tangle solutions with three-dimensional models of the complexes, we propose a recombination mechanism in which the synaptic intermediate is characterized by a non-planar, possibly near-tetrahedral, Holliday-junction intermediate. Only modest conformational changes within this structure are needed to form the symmetric, planar DNA junction, which may be characteristic of shorter-lived intermediates along the recombination pathway.     

Evaluation of elastic properties of atomistic DNA models

A number of intriguing aspects in dynamics of double-helical DNA is related to the coupling between its macroscopic and microscopic states. A link between the elastic properties of long DNA chains and their atom-level dynamics can be established by comparing the worm-like chain model of polymer DNA with the conformational ensembles produced by molecular dynamics simulations. This problem is complicated by the complexity of the DNA structure, the small size of DNA fragments, and relatively short trajectory durations accessible in computer simulations of microscopic DNA dynamics. A careful study of all these aspects has been performed by using longer DNA fragments and increased durations of MD trajectories as compared to earlier such investigations. Special attention is paid to the necessary conditions and criteria of time convergence, and the possibility to increase the sampling by using constrained DNA models and simplified simulation conditions. It is found that dynamics of 25-mer duplexes with regular sequences agrees well with the worm-like chain theory and that accurate evaluation of DNA elastic parameters requires at least two turns of the double helix and approximately 20-ns duration of trajectories. Bond length and bond-angle constraints affect the estimates within numerical errors. In contrast, simplified treatment of solvation can strongly change the observed elastic parameters of DNA. The elastic parameters evaluated for AT- and GC-alternating duplexes reasonably agree with experimental data and suggest that, in different basepair sequences, the torsional and stretching elasticities vary stronger than the bending stiffness.     

Structural aspects of RecA-dependent homologous strand exchange involving human telomeric DNA

Telomeric DNA sequences in human cells and those of other vertebrates consist of long d(TTAGGG) repeats. In somatic cells, telomeres shorten every cell division with shortening serving as a mitotic clock that counts cell divisions and ultimately results in cellular senescence. Telomere length is principally maintained by a ribonucleoprotein, telomerase. However, a non-negligible proportion of human cells use a recombination-based mechanism for telomere maintenance, termed alternative maintenance of telomeres (ALT). Although the molecular mechanism of ALT is not known, GT-rich sequences in prokaryotes and eukaryotes display high levels of recombination relative to those of non-GT-rich DNA. We show that human telomeric strand-exchange complexes mediated by Escherichia coli RecA protein differ from those formed with nontelomeric sequences. Moreover, telomeric strand-exchange intermediates, unlike those involving nontelomeric sequences, exhibit a tendency to form higher-order nucleoprotein structures. We propose that the strong DNA unwinding activity inherent in the assembly of the RecA strand-exchange complex promotes the formation of alternative DNA structures at human telomeric loci. Organization of these noncanonical structures into higher-order complexes involving multiple DNA duplexes could facilitate the search for homology on different DNA molecules and provide a framework for understanding recombination-dependent mechanisms of telomere maintenance.     

Effects of the nucleoid protein HU on the structure, flexibility, and ring-closure properties of DNA deduced from Monte Carlo simulations

The histone-like HU (heat unstable) protein plays a key role in the organization and regulation of the Escherichia coli genome. The nonspecific nature of HU binding to DNA complicates analysis of the mechanism by which the protein contributes to the looping of DNA. Conventional models of the looping of HU-bound duplexes attribute the changes in biophysical properties of DNA brought about by the random binding of protein to changes in the effective parameters of an ideal helical wormlike chain. Here, we introduce a novel Monte Carlo approach to study the effects of nonspecific HU binding on the configurational properties of DNA directly. We randomly decorated segments of an ideal double-helical DNA with HU molecules that induce the bends and other structural distortions of the double helix find in currently available X-ray structures. We find that the presence of HU at levels approximating those found in the cell reduces the persistence length by roughly threefold compared with that of naked DNA. The binding of protein has particularly striking effects on the cyclization properties of short duplexes, altering the dependence of ring closure on chain length in a way that cannot be mimicked by a simple wormlike model and accumulating at higher-than-expected levels on successfully closed chains. Moreover, the uptake of protein on small minicircles depends on chain length, taking advantage of the HU-induced deformations of DNA structure to facilitate ligation. Circular duplexes with bound HU show much greater propensity than protein-free DNA to exist as negatively supercoiled topoisomers, suggesting a potential role of HU in organizing the bacterial nucleoid. The local bending and undertwisting of DNA by HU, in combination with the number of bound proteins, provide a structural rationale for the condensation of DNA and the observed expression levels of reporter genes in vivo.