Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.     

Assembly and characterization of five-arm and six-arm DNA branched junctions.

DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.     

Structure of Three-Way DNA Junctions 1. Non-Planar DNA Geometry

Abstract

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20–21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60–90°. Rotation of the hairpin position in the three- way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60–90° between the arms. Computer modeling of the three-way junction with 60° pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.     

Intramolecular transposition by Tn10

Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.     

Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase

Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pairs of single strands at a time. The product of the first exchange reaction is a four-way DNA junction, the Holliday intermediate. The conformational dynamics by which the recombination complex "isomerizes" from the Holliday-forming to the Holliday-resolving mode are not well understood. Experiments with the lambda Int and Escherichia coli XerC/XerD systems imply that the strand configurations at the branch point of the protein-free junction dictate the resolution mode in the protein-bound junction. We have examined the question of strand bias during resolution for the Flp system by using a series of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering. We have not observed a strong resolution bias in favor of the strands designed to assume the "crossed" configuration within the unbound junction. The resolution patterns with antiparallel junctions in a variety of substrate contexts reveal either parity in strand choice, or only modest disparity. On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be explained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinants at the tethered ends.     

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.     

Impact of the third-strand orientation on the thermodynamic stability of the four-way DNA junction

The physical properties of a triple-helical DNA four-way junction J(T2T4) have been characterized by means of UV spectroscopy, CD spectroscopy, and differential scanning calorimetry (DSC). J(T2T4) is another four-way junction that was designed in addition to J(T1T3) (N. Makube and H. H. Klump (2000) Arch. Biochem. Biophys. 377, 31-42) to study the effects of third strands on the stability of the four-way junction with triple-helical arms. The pH titration curves illustrate the sequential folding of single strands to double-helical four-way junctions and finally the binding of third strands to their respective W-C duplexes. CD measurements confirm triplex formation under appropriate pH and ionic strength conditions. The CD spectra also suggest different melting patterns for the triple-helical arms of J(T2T4). The melting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium concentration and low pH conditions enhances and stabilizes the overall structure of the junction. The results also indicate that all triplexes in J(T2T4) are formed in the absence of salt and at low pH; however, the junction may, under these conditions, assume a conformation different from the one assumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions were determined. The loops are designed to have the same enthalpic effect on the different arms. The stabilizing effect of the loops is more pronounced when those loops are shifted from arms 1 and 3 in J(T1T3) to arms 2 and 4 in J(T2T4) without changing any of the sequences. Overall, J(T2T4) is slightly more stable than J(T1T3). The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that binding of the third strand in either of the two orientations 5'5'3' (J(T2T4)) or 5'3'3' (J(T1T3)) stabilizes the underlying double-helical four-way junction and its triple-helical arms.     

An archaeal Holliday junction resolving enzyme from Sulfolobus solfataricus exhibits unique properties

The rearrangement and repair of DNA by homologous recombination often involves the creation of Holliday junctions, which must be cleaved by junction-specific endonucleases to yield recombinant duplex DNA products. Holliday junction resolving enzymes are a ubiquitous class of proteins with diverse structural and mechanistic characteristics. We have characterised an endonuclease (Hje) from the thermophilic crenarchaeote Sulfolobus solfataricus that exhibits a high degree of specificity for Holliday junctions via an apparently novel mechanism. Hje resolves four-way DNA junctions by the introduction of paired nicks in a reaction that is independent of the local nucleotide sequence, but is restricted solely to strands that are continuous in the stacked-X form of the junction. Three-way DNA junctions are cleaved only when the presence of a bulge in one strand allows the junction to stack in an analogous manner to four-way junctions. These properties differentiate Hje from all other known junction resolving enzymes.     

Resolution of undistorted symmetric immobile DNA junctions by vaccinia topoisomerase I

Holliday junctions are intermediates in genetic recombination. They consist of four strands of DNA that flank a branch point. In natural systems, their sequences have 2-fold (homologous) sequence symmetry. This symmetry enables the molecules to undergo an isomerization, known as branch migration, that relocates the site of the branch point. Branch migration leads to polydispersity, which makes it difficult to characterize the physical properties of the junction and the effects of the sequence context flanking the branch point. Previous studies have reported two symmetric junctions that do not branch migrate: one that is immobilized by coupling to an asymmetric junction in a double crossover context, and a second that is based on molecules containing 5',5' and 3',3' linkages. Both are flawed by distorting the structure of the symmetric junction from its natural conformation. Here, we report an undistorted symmetric immobile junction based on the use of DNA parallelogram structures. We have used a series of these junctions to characterize the junction resolution reaction catalyzed by vaccinia virus DNA topoisomerase. The resolution reaction entails cleavage and rejoining at CCCTT/N recognition sites arrayed on opposing sides of the four-arm junction. We find that resolution is optimal when the scissile phosphodiester (Tp/N) is located two nucleotides 5' to the branch point on the helical strand. Covalent topoisomerase-DNA adducts are precursors to recombinant strands in all reactions, as expected. Kinetic analysis suggests a rate limiting step after the first-strand cleavage.     

Cruciform-resolvase interactions in supercoiled DNA

T4 endonuclease VII, which cleaves Holliday-like junctions in DNA, specifically cleaves short inverted repeats in supercoiled plasmids. These sequences are subject to site-specific cleavage by single-strand-specific nucleases, and cruciform formation has been suggested as an explanation for this observation. This proposal is greatly strengthened by the present data, since a formal analogy between cruciform structures and Holliday junctions exists. Resolution of a variety of unrelated cruciform sequences demonstrates that the cleavage process results in a linear molecule with hairpin ends and single ligatable nicks at positions corresponding to the stem-base of the cruciform. In two examples mapped in detail, the cleavages are exclusively introduced at two or three nucleotides from the end of the symmetric sequence at the 5' side on each strand. These studies demonstrate the potential of endonuclease VII as a probe of cruciform structure and the utility of short cruciform structures as Holliday junction models.     

Construction of a synthetic Holliday junction analog and characterization of its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions

We describe the construction and characterization of an oligonucleotide Holliday junction analog and characterize its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions. A Holliday junction analog containing four duplex arms and 54 base pairs was constructed by annealing four unique synthetic oligonucleotides. Mixing curve analysis showed that the complex contained a 1:1:1:1 mol ratio of the four unique sequence strands. In addition, a linear duplex with a sequence identical to two of the junction arms was also constructed for use as a control fragment. High resolution gel exclusion chromatography was used to purify and characterize the synthetic junction. The synthetic Holliday junction was found to be a specific inhibitor of a S. cerevisiae enzyme that catalyzes the cleavage of Holliday junctions. Under standard cleavage conditions, 50% inhibition was observed at a synthetic Holliday junction to substrate ratio of 7/1, whereas no inhibition by linear duplex was observed at molar ratios in excess of 150/1. Kinetic analysis showed that Holliday junction was a competitive inhibitor of the reaction and had an apparent Ki = 2.5 nM, although the mode of inhibition was complex. The synthetic Holliday junction was not a substrate for the enzyme, but was found to form a specific complex with the enzyme as evidenced by polyacrylamide gel electrophoresis DNA binding assays.     

Stability and structure of three-way DNA junctions containing unpaired nucleotides.

Non-paired nucleotides stabilize the formation of three-way helical DNA junctions. Two or more unpaired nucleotides located in the junction region enable oligomers ten to fifteen nucleotides long to assemble, forming conformationally homogeneous junctions, as judged by native gel electrophoresis. The unpaired bases can be present on the same strand or on two different strands. Up to five extra bases on one strand have been tested and found to produce stable junctions. The formation of stable structures is favored by the presence of a divalent cation such as magnesium and by high monovalent salt concentration. The order-disorder transition of representative three-way junctions was monitored optically in the ultraviolet and analyzed to quantify thermodynamically the stabilization provided by unpaired bases in the junction region. We report the first measurements of the thermodynamics of adding an unpaired nucleotide to a nucleic acid three-way junction. We find that delta delta G degrees (37 degrees C) = +0.5 kcal/mol for increasing the number of unpaired adenosines from two to three. Three-way junctions having reporter arms 40 base-pairs long were also prepared. Each of the three reporter arms contained a unique restriction site 15 base-pairs from the junction. Asymmetric complexes produced by selectively cleaving each arm were analyzed on native gels. Cleavage of the double helical arm opposite the strand having the two extra adenosines resulted in a complex that migrated more slowly than complexes produced by cleavage at either of the other two arms. It is likely that the strand containing the unpaired adenosines is kinked at an acute angle, forming a Y-shaped, rather than a T-shaped junction.     

Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure.

Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.     

Substrate specificity of RusA resolvase reveals the DNA structures targeted by RuvAB and RecG in vivo

RusA endonuclease cleaves Holliday junctions by introducing paired strand incisions 5' to CC dinucleotides. Coordinated catalysis is achieved when both subunits of the homodimer interact simultaneously with cleavage sites located symmetrically. This requirement confers Holliday junction specificity. Uncoupled catalysis occurs when binding interactions are disturbed. Genetic studies indicate that uncoupling occurs rarely in vivo, and DNA cleavage is therefore restricted to Holliday junctions. We exploited the specificity of RusA to identify the DNA substrates targeted by the RuvAB and RecG branch-migration proteins in vivo. We present evidence that replication restart in UV-irradiated cells relies on the processing of stalled replication forks by RecG helicase and of Holliday junctions by the RuvABC resolvasome, and that RuvAB alone may not promote repair.     

Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4

The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template.     

The repair of psoralen monoadducts by the Escherichia coli UvrABC endonuclease.

We have examined the interactions of UvrABC endonuclease with DNA containing the monoadducts of 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP). The UvrA and UvrB proteins were found to form a stable complex on DNA that contains the psoralen monoadducts. Subsequent binding of UvrC protein to this complex activates the UvrABC endonuclease activity. As in the case of incision at pyrimidine dimers, a stable protein-DNA complex was observed after the incision events. For both 8-MOP and TMP, the UvrABC endonuclease incised the monoadduct-containing strand of DNA on the two sides of the monoadduct with 12 bases included between the two cuts. One incision was at the 8th phosphodiester bond on the 5' side of the modified base. The other incision was at the 5th phosphodiester bond 3' to the modified base. The UvrABC endonuclease incision data revealed that the reactivity of psoralens is 5'TpA greater than 5'ApT greater than 5'TpG.     

The tertiary structure of the four-way DNA junction affords protection against DNase I cleavage.

The accessibility of phosphodiester bonds in the DNA of four-way helical junctions has been probed with the nuclease DNase I. Regions of protection were observed on all four strands of the junctions, that tended to be longer on the strands that are exchanged between the coaxially stacked pairs of helices. The protected regions on the continuous strands of the stacked helices were not located exactly at the junction, but were displaced towards the 3' side of the strand. This is the region of backbone that becomes located in the major groove of the opposed helix in the non-crossed, right-handed structure for the junction, and might therefore be predicted to be protected against cleavage by an enzyme. However, the major grooves of the structure remain accessible to the much smaller probe dimethyl sulphate.     

No braiding of Holliday junctions in positively supercoiled DNA molecules

The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.     

Sequence of the genome ends and of the junction between the ends in concatemeric DNA of pseudorabies virus.

The nucleotide sequences of the termini of the mature pseudorabies virus genome and of the junction between these termini in concatemeric DNA were compared. To ensure conservation of unmodified 5' and 3' termini, the end fragments obtained directly (uncloned) from mature viral DNA were sequenced. The sequence obtained from 5' and 3' end labeling revealed that whereas the L terminus was blunt ended, the S terminus had a 2-base (GG) 3' overhang. The sequences spanning the junction between the termini present in concatemeric DNA was also determined and compared with that expected when the two ends of the mature DNA were juxtaposed. This comparison showed that in concatemeric DNA the ends of the mature genome had become joined by blunt-end ligation of one of the strands and that the 2-nucleotide gap on the other strand had been repaired. A significant degree of homology between the sequences spanning the junction between the ends of the varicella-zoster virus and pseudorabies virus genomes was found.     

Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.