RecA protein promoted homologous pairing in vitro. Pairing between linear duplex DNA bound to HU Protein (nucleosome cores) and nucleoprotein filaments of recA protein-single-stranded DNA

RecA protein promotes two distinct types of synaptic structures between circular single strands and duplex DNA; paranemic joints, where true intertwining of paired strands is prohibited and the classically intertwined plectonemic form of heteroduplex DNA. Paranemic joints are less stable than plectonemic joints and are believed to be the precursors for the formation of plectonemic joints. We present evidence that under strand exchange conditions the binding of HU protein, from Escherichia coli, to duplex DNA differentially affects homologous pairing in vitro. This conclusion is based on the observation that the formation of paranemic joint molecules was not affected, whereas the formation of plectonemic joint molecules was inhibited from the start of the reaction. Furthermore, introduction of HU protein into an ongoing reaction stalls further increase in the rate of the reaction. By contrast, binding of HU protein to circular single strands has neither stimulatory nor inhibitory effect. Since the formation of paranemic joint molecules is believed to generate positive supercoiling in the duplex DNA, we have examined the ability of positive superhelical DNA to serve as a template in the formation of paranemic joint molecules. The inert positively supercoiled DNA could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. Taken collectively, these results indicate that the structural features of the bacterial chromosome which include DNA supercoiling and organization of DNA into nucleosome-like structures by HU protein modulate homologous pairing promoted by the nucleoprotein filaments of recA protein single-stranded DNA.     

On RecA protein-mediated homologous alignment of two DNA molecules. Three strands versus four strands

The recA protein from Escherichia coli can homologously align two duplex DNA molecules; however, this interaction is much less efficient than the alignment of a single strand and a duplex. Three strand paranemic joints are readily detected. In contrast, duplex-duplex pairing is detected only when the incoming (second) duplex is negatively supercoiled, and even here the pairing is inefficient. The recA protein-promoted four strand exchange reaction is initiated in a three strand region, with efficiency increasing with the length of potential three strand pairing available for initiation. This indicates that a paranemic joint involving three DNA strands may be an important intermediate in all recA protein-mediated DNA strand exchange reactions and that the presence of three strands rather than four is a fundamental structural parameter of paranemic joints.     

Thermodynamics of Forming a Parallel DNA Crossover

The process of genetic recombination involves the formation of branched four-stranded DNA structures known as Holliday junctions. The Holliday junction is known to have an antiparallel orientation of its helices, i.e., the crossover occurs between strands of opposite polarity. Some intermediates in this process are known to involve two crossover sites, and these may involve crossovers between strands of identical polarity. Surprisingly, if a crossover occurs at every possible juxtaposition of backbones between parallel DNA double helices, the molecules form a paranemic structure with two helical domains, known as PX-DNA. Model PX-DNA molecules can be constructed from a variety of DNA molecules with five nucleotide pairs in the minor groove and six, seven or eight nucleotide pairs in the major groove. A topoisomer of the PX motif is the juxtaposed JX₁ molecule, wherein one crossover is missing between the two helical domains. The JX₁ molecule offers an outstanding baseline molecule with which to compare the PX molecule, so as to measure the thermodynamic cost of forming a crossover in a parallel molecule. We have made these measurements using calorimetric and ultraviolet hypochromicity methods, as well as denaturing gradient gel electrophoretic methods. The results suggest that in relaxed conditions, a system that meets the pairing requirements for PX-DNA would prefer to form the PX motif relative to juxtaposed molecules, particularly for the 6:5 structure.     

The formation of plectonemic joints by the recA protein of Escherichia coli. Requirement for ATP hydrolysis

Formation of D-loops during the exchange of strands between a circular single-stranded DNA and a completely homologous linear duplex proceeds optimally when the duplex DNA is added to the complex of recA protein and single-stranded DNA formed in the presence of single-stranded DNA-binding protein and ATP. D-loops are undetectable when 200 microM adenosine 5'-O-(thiotriphosphate) is substituted for ATP. D-loops can be formed in the presence of adenosine 5'-O-(thiotriphosphate) if recA protein is the last component added to the reaction. However, these D-loops, which depend upon homologous sequences, are unstable upon deproteinization and are formed to a more limited extent than the structures formed with ATP. This finding indicates that D-loops formed under these conditions may be largely nonintertwined paranemic structures rather than plectonemic structures in which two of the strands are interwoven. When adenosine 5'-O-(thiotriphosphate) is added to an ongoing reaction containing ATP, formation of plectonemic structures and ATP hydrolysis is inhibited to an equivalent extent. We, therefore, conclude that ATP hydrolysis is required for the formation of plectonemic structures.     

Inhibition of DNA synthesis by aphidicolin induces supercoiling in simian virus 40 replicative intermediates.

Highly torsionally stressed replicative intermediate SV40 DNA molecules are produced when ongoing replicative DNA synthesis is inhibited by aphidicolin, a specific inhibitor of DNA polymerase alpha. The high negative superhelical density of these molecules can be partially released by intercalating drugs such as chloroquine or ethidium bromide. The torsionally stressed replicative intermediates bind to monoclonal anti-Z-DNA antibodies. Electron microscopy of anti-Z-DNA cross-linked to torsionally stressed replicative intermediates shows that the antibody specifically binds close to the replication forks. The superhelical structures are not formed when SV40 DNA replication is inhibited by both aphidicolin and novobiocin, suggesting that a topoisomerase type II-like enzyme is somehow involved in the introduction of torsional strain in replicative intermediate DNA. One interpretation of our data is that fork movement continues to some rather limited extent when SV40 DNA synthesis in replicative chromatin is blocked by aphidicolin. After deproteinization, the exposed single-stranded DNA branches reassociate to form paranemic DNA structures with left-handed helical stretches, while the reduced linking number of the parental strands induces a high negative superhelical density.     

Mechanism of meiotic recombination in eukaryotes—A theory

It is proposed that in meiotic chromosomes single strand breaks of DNA originate either in the delayed regions of replicons or as a result of the excision activity of DNA polymerase during zygotene DNA synthesis. Rejoining of the break points belonging to non-sister chromatids takes place by switching over of the polymerase from one strand of DNA to another non-sister strand of the same polarity and gives rise to recombination intermediates (half-chromatid chiasmata). Strand migration in a recombination intermediate or copying of the same parental strand twice during zygotene as a consequence of a delay in copying the homologous strand would lead to gene conversion. Nicking of the cross strands (parental strands) in any recombination intermediate and subsequent repair leads to recombination for flanking markers. A possible way in which three-strand double crossovers occur and the process of recombination are discussed.     

Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products.     

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA.

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.     

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.     

Synapsis and the formation of paranemic joints by E. coli RecA protein

E. coli RecA protein promotes the homologous pairing of a single strand with duplex DNA even when certain features of the substrates, such as circularity, prohibit the true intertwining of the newly paired strands. The formation of such nonintertwined or paranemic joints does not require superhelicity, and indeed can occur with relaxed closed circular DNA. E. coli topoisomerase I can intertwine the incoming single strand in the paranemic joint with its complement, thereby topologically linking single-stranded DNA to all of the duplex molecules in the reaction mixture. The efficiency of formation of paranemic joints, the time course, and estimates of their length, all suggest that they represent true synaptic intermediates in the pairing reaction promoted by RecA protein.     

Synapsis promoted by Ustilago rec1 protein

Ustilago rec 1 protein pairs homologous DNA molecules by promoting both synapsis and strand transfer. Complexes formed with rec 1 protein and a homologous combination of single-stranded and duplex DNA that appear to be synaptic structures can be detected by use of a nitrocellulose filter assay. The nascent heteroduplex formed during synapsis is a paranemic joint in which the single-stranded DNA pairs, but does not interwind, with its complement in the duplex molecule. Formation of the paranemic joint is accompanied by duplex unwinding and genesis of left-handed Z-DNA.     

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses I. Directing Influence of DNA in the Reaction

The template requirements and deoxyribonucleic acid (DNA) products of the DNA polymerases isolated from Rauscher leukemia and avian myeloblastosis viruses have been examined. All DNA preparations or synthetic polydeoxynucleotides which are active as primers possess a duplex structure containing single-stranded regions with a 3'-hydroxyl terminus. Native DNA and fully single-stranded DNA are inactive; moreover, their activity is not enhanced by sonic oscillation or treatment with micrococcal nuclease, Neurospora nuclease, or low levels of deoxyribonuclease I. Poor DNA templates are activated by treatment with exonuclease III, large amounts of deoxyribonuclease I, or an endonuclease isolated from Rauscher viral preparations. In reactions primed with deoxyadenylate-deoxythymidylate copolymer, the product formed is covalently attached to primer strands, indicating that no new strands are initiated. DNA polymerase products formed with exonuclease III- or deoxyribonuclase I-treated DNA are duplex structures. Short single-stranded regions are completely filled in, whereas long single-stranded regions are only partly repaired. DNA preparations containing extensive single-stranded regions are poorly utilized as templates.     

Three-stranded paranemic joints: architecture, topological constraints and movement

The RecA and SSB proteins will catalyze the joining of two DNA molecules containing homologous sequences but lacking homologous ends in a reaction termed paranemic joining. The absence of homologous ends can be achieved by (1) pairing two circular DNAs or (2) using linear DNA(s) with ends lacking homology to the pairing partner. Here we have used electron microscopy (EM) to examine such pairings. Circular M13 single-stranded (ss) DNA enveloped by RecA protein into a presynaptic filament was paired with linear M13mp7 double-stranded (ds) DNA containing non-M13 sequences at its ends. Joint complexes were frequently seen in which the dsDNA was joined with the presynaptic filament over several kilobase (10(3) bases) lengths of the dsDNA. In this region, the presynaptic filament appeared disorganized as contrasted to the customary helical structure of the filament containing only a single strand of DNA. The same ultrastructure, but with greater detail, was observed when the samples were prepared for EM without fixation using a new method of fast-freezing and freeze-drying. EM immunogold staining demonstrated the presence of SSB protein in the disorganized region containing all three strands, but not in the regular helically arranged region. Psoralen photo-crosslinking of the DNA in the joint complexes revealed that the three DNA strands were in close proximity only over a single short (200 to 300 base-pairs) region. The joining of nicked circular M13 dsDNA and presynaptic filaments containing circular M13 ssDNA resulted in the intertwining of the dsDNA about the circular presynaptic filament. The joints produced in this case were short, as was the single region of psoralen photo-crosslinking of the three DNA strands. A model of how these long three-stranded joints form is presented involving the movement of a short "true" paranemic joint along the presynaptic filament.     

Nature of the single-stranded DNA in replicating adenovirus type 5 DNA.

Single-stranded regions in replicating adenovirus type 5 DNA were isolated and hybridized in solution to the separated strands of adenovirus 2 or 5 DNA. The results showed that the two strands of adenovirus 5 DNA are exposed to almost the same extent during replication, suggesting that displacement synthesis may start from either end of the viral DNA.     

Three-stranded DNA structure; is this the secret of DNA homologous recognition?

A novel type of triple-stranded DNA structure was proposed by several groups to play a crucial role in homologous recognition between single- and double-stranded DNA molecules. In this still putative structure a duplex DNA was proposed to co-ordinate a homologous single strand in its major groove side. In contrast to the well-characterized pyrimidine-purine-pyrimidine triplexes in which the two like strands are antiparallel and which are restricted to poly-pyrimidine-containing stretches, the homology-specific triplexes would have like strands in parallel orientation and would not be restricted to any particular sequence provided that there is a homology between interacting DNA molecules. For many years the stereo-chemical possibility of forming homology-dependent three- or four-stranded DNA structures during the pairing stage of recombination reactions was seriously considered in published papers. However, only recently has there been a marked increase in the number of papers that have directly tested the formation of triple-stranded DNA structures during the actual pairing stage of the recombination reaction. Unfortunately the results of these tests are not totally clear cut; while some laboratories presented experimental evidence consistent with the formation of triplexes, others studying the same or very similar systems offered alternative explanations. The aim of this review is to present the current state of the central question in the mechanism of homologous recombination, namely, what kind of DNA structure is responsible for DNA homologous recognition. Is it a novel triplex structure or just a classical duplex?     

The stretched DNA geometry of recombination and repair nucleoprotein filaments

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.     

Reassociation rate limited displacement of DNA strands by branch migration.

Large branched DNA structures are constructed by two-step reassociation of separated complementary strands from restriction fragments of different lengths. The displacement of DNA strands initially annealed to longer complementary DNA sequences, a process mediated by branch migration, is very rapid and has thus far been followed only under conditions which are second order, DNA reassociation rate limiting. The average lifetime of branched DNA leading to displacement of 1.6 Kb strands is estimated to be less than 10 seconds under conditions of DNA reassociation, Tm-25 degrees C. Several DNA-binding drugs, including intercalating dyes, have been tested to determine their influence, if any, on the kinetics of DNA strand displacements by branch migration. Only actinomycin D was found to have significant effect under the conditions we have described. The kinetics of the strand displacement in the presence of low concentrations of actinomycin D remain second order and slower rate of strand displacement must be attributed to decreased rate of reassociation of DNA strands to form the branched intermediates. Consideration is given to the potential manipulation of DNA structures at site-directed branches and the limitations due to rapid strand displacements. The feasibility of constructing sufficiently large branched DNA regions to approach first order, branch migration rate limiting kinetics is also discussed.     

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.     

recF-dependent and recF recB-independent DNA gap-filling repair processes transfer dimer-containing parental strands to daughter strands in Escherichia coli K-12 uvrB.

The processes for repairing DNA daughter-strand gaps were studied in UV-irradiated uvrB, uvrB recB, uvrB recF, and uvrB recB recF cells of Escherichia coli K-12. The dimer-containing parental DNA was found to be joined to daughter strands during postreplication repair in all four strains examined. Therefore, both the major (recF-dependent) and the minor (recF recB-independent) gap-filling processes repair DNA daughter-strand gaps by transferring parental strands into daughter strands.