T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs.

We have formed four-arm branched DNA junctions that contain no more than a single base pair of branch migratory freedom. Recently, we have shown that these Holliday junction analogs have twofold symmetric protection patterns in solution when probed with hydroxyl radicals: two opposite strands of one junction show extensive protection near the branch point, while the other pair of opposite strands is virtually as susceptible as a double helix. In a different junction, the hydroxyl radical protection pattern is reversed. These patterns suggest that a crossover-isomer bias exists in these molecules and that the protected strands form the crossover between helices. Here, we examine the cleavage pattern of these structures when they are resolved by T4 endonuclease VII. Junctions are formed from a single shamrock-shaped molecule, which contains 5', 3', or internal labels. The enzyme shows a preference for resolving these modified junctions at sites near those protected from hydroxyl radicals. This result suggests that only crossover strands in a Holliday junction are cleaved, and thus an odd number of crossover isomerizations must occur when flanking markers are exchanged.     

The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions

Holliday junctions are important structural intermediates in recombination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGGTACCGG) as a Holliday junction at the nominal resolution of 2. 1 A. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson-Crick base pairs; it therefore represents the intermediate proposed by Holliday as being involved in homologous recombination. The junction is in the stacked-X conformation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4 degrees as a right-handed X. A sequence comparison with previous B-DNA and junction crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3'-C x G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the phosphates at the crossover strands. Interactions within this core define the conformation of the Holliday junction, including the angle relating the stacked duplexes and how the base pairs are stacked in the stable form of the junction.     

Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.     

RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands.

The RuvC protein of Escherichia coli resolves Holliday junctions during genetic recombination and the postreplicational repair of DNA damage. Using synthetic Holliday junctions that are constrained to adopt defined isomeric configurations, we show that resolution occurs by symmetric cleavage of the continuous (noncrossing) pair of DNA strands. This result contrasts with that observed with phage T4 endonuclease VII, which cleaves the pair of crossing strands. In the presence of RuvC, the pair of continuous strands (i.e., the target strands for cleavage) exhibit a hypersensitivity to hydroxyl radicals. These results indicate that the continuous strands are distorted within the RuvC/Holliday junction complex and that RuvC-mediated resolution events require protein-directed structural changes to the four-way junction.     

A Holliday recombination intermediate is twofold symmetric.

Four-arm Holliday structures are ephemeral intermediates in genetic recombination. We have used an oligodeoxynucleotide system to form immobile DNA junctions, which are stable analogs of Holliday structures. We have probed the equilibrium structure of a junction by means of hydroxyl radicals generated by the reaction of iron(II)EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern shows twofold symmetry throughout the molecule. Strong protection from hydroxyl radical attack is evident on two strands near the branch site, and weaker protection may be seen four or five residues 3' to the branch site on the other two strands. No other position appears significantly distinct from double-helical DNA controls. From these data, we conclude that the Holliday junction is a twofold symmetric complex whose four arms form two stacking domains.     

Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions.

An enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions was partially purified approximately 500- to 1000-fold by DEAE-cellulose chromatography, gel filtration on Sephacryl S300, and chromatography on single-stranded DNA-cellulose. The partially purified enzyme did not have any detectable nuclease activity when tested with single-stranded or double-stranded bacteriophage T7 substrate DNA and did not have detectable endonuclease activity when tested with bacteriophage M13 viral DNA or plasmid pBR322 covalently closed circular DNA. Analysis of the products of the cruciform cleavage reaction by electrophoresis on polyacrylamide gels under denaturing conditions revealed that the cruciform structure was cleaved at either of two sites present in the stem of the cruciform and was not cleaved at the end of the stem. The cruciform cleavage enzyme was able to cleave the Holliday junction present in bacteriophage G4 figure-8 molecules. Eighty percent of these Holliday junctions were cleaved in the proper orientation to generate intact chromosomes during genetic recombination.     

Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer

In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins. Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration. We solved the crystal structure of the Holliday junction bound to a single Escherichia coli RuvA tetramer at 3.1-A resolution. In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center. The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry. Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit. The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding.     

Observing spontaneous branch migration of Holliday junctions one step at a time

Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.     

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.     

Single-molecule study of RuvAB-mediated Holliday-junction migration

Branch migration of Holliday junctions is an important step of genetic recombination and DNA repair. In Escherichia coli, this process is driven by the RuvAB complex acting as a molecular motor. Using magnetic tweezers, we studied the RuvAB-directed migration of individual Holliday junctions formed between two approximately 6-kb DNA molecules of identical sequence, and we measured the migration rate at 37 degrees C and 1 mM ATP. We directly demonstrate that RuvAB is a highly processive DNA motor protein that is able to drive continuous and unidirectional branch migration of Holliday junctions at a well defined average speed over several kilobases through homologous sequences. We observed directional inversions of the migration at the DNA molecule boundaries leading to forth-and-back migration of the branch point and allowing us to measure the migration rate in the presence of negative or positive loads. The average migration rate at zero load was found to be approximately 43 bp/sec. Furthermore, the load dependence of the migration rate is small, within the force range of -3.4 pN (hindering force) to +3.4 pN (assisting force).     

A Holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea.

The Holliday junction is an essential intermediate of homologous recombination. RecA of Bacteria, Rad51 of Eukarya, and RadA of Archaea are structural and functional homologs. These proteins play a pivotal role in the formation of Holliday junctions from two homologous DNA duplexes. RuvC is a specific endonuclease that resolves Holliday junctions in Bacteria. A Holliday junction-resolving activity has been found in both yeast and mammalian cells. To examine whether the paradigm of homologous recombination apply to Archaea, we assayed and found the activity to resolve a synthetic Holliday junction in crude extract of Pyrococcus furiosus cells. The gene, hjc (Holliday junction cleavage), encodes a protein composed of 123 amino acids, whose sequence is not similar to that of any proteins with known function. However, all four archaea, whose total genome sequences have been published, have the homologous genes. The purified Hjc protein cleaved the recombination intermediates formed by RecA in vitro. These results support the notion that the formation and resolution of Holliday junction is the common mechanism of homologous recombination in the three domains of life.     

Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.     

Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.

The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[gamma-thio]triphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coli.     

Correction of proliferation and drug sensitivity defects in the progeroid Werner's Syndrome by Holliday junction resolution

The progeroid Werner's syndrome (WS) represents the best current model of human aging. It is caused by loss of the WRN helicase/exonuclease, resulting in high levels of replication fork stalling and genomic instability. Current models suggest that characteristic WS phenotypes of poor S phase progression, low proliferative capacity, and drug hypersensitivity are the result of accumulation of alternative DNA structures at stalled or collapsed forks during DNA replication, and Holliday junction resolution has been shown to enhance survival of cis-platin-treated WS cells. Here, we present a direct test of the hypothesis that the replication/repair defect in unstressed WS cells is the result of an inability to resolve recombination intermediates. We have created isogenic WS cell lines expressing a nuclear-targeted bacterial Holliday junction endonuclease, RusA, and show that Holliday junction resolution by RusA restores DNA replication capacity in primary WS fibroblasts and enhances their proliferation. Furthermore, RusA expression rescues WS fibroblast hypersensitivity to replication fork blocking agents camptothecin and 4NQO, suggesting that the hypersensitivity is caused by inappropriate recombination at DNA structures formed when the replication fork arrests or collapses at 4NQO- or camptothecin-induced lesions. This work is the first to demonstrate that Holliday junction accumulation in primary Werner syndrome fibroblasts results in their poor proliferative capacity, and to rescue WS hypersensitivity to camptothecin and 4NQO by Holliday junction resolution.     

RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro.

The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity. The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.     

The kinetics of spontaneous DNA branch migration.

An important step in genetic recombination is DNA branch migration, the movement of the Holliday junction or exchange point between two homologous duplex DNAs. We have determined kinetic parameters of spontaneous branch migration as a function of temperature and ionic conditions. The branch migration substrates consist of two homologous duplex DNAs each having two single-strand tails at one end that are complementary to the corresponding single-strand tails of the other duplex. Upon rapid annealing of the two duplex DNAs, a four-stranded intermediate is formed that has a Holliday junction at one end of the duplexes. Branch migration to the opposite end of the duplexes results in complete strand exchange and formation of two duplex products. The rate of branch migration is exceedingly sensitive to the type of metal ions present. In magnesium, branch migration is quite slow with a step time, tau, equal to 300 msec at 37 degrees C. Surprisingly, branch migration in the absence of magnesium was 1000 times faster. Despite this difference in rates, apparent activation energies for the branch migration step in the presence and absence of magnesium are similar. Since metal ions have a profound effect on the structure of the Holliday junction, it appears that the structure of the branch point plays a key role in determining the rate of spontaneous DNA branch migration. We discuss the role of proteins in promoting the branch migration step during homologous recombination.     

Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.     

Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions

Holliday junctions form during DNA repair and homologous recombination processes. These processes entail branch migration, whereby the length of two arms of a cruciform increases at the expense of the two others. Branch migration is carried out in prokaryotic cells by the RuvAB motor complex. We study RuvAB-catalyzed branch migration by following the motion of a small paramagnetic bead tethered to a surface by two opposing arms of a single cruciform. The bead, pulled under the action of magnetic tweezers, exerts tension on the cruciform, which in turn transmits the force to a single RuvAB complex bound at the crossover point. This setup provides a unique means of measuring several kinetic parameters of interest such as the translocation rate, the processivity, and the force on the substrate against which the RuvAB complex cannot effect translocation. RuvAB-catalyzed branch migration proceeds with a small, discrete number of rates, supporting the view that the monomers comprising the RuvB hexameric rings are not functionally homogeneous and that dimers or trimers constitute the active subunits. The most frequently encountered rate, 98 +/- 3 bp/sec, is approximately five times faster than previously estimated. The apparent processivity of branch migration between pauses of inactivity is approximately 7,000 bp. Branch migration persists against opposing forces up to 23 pN.     

In vivo cloning of artificial DNA nanostructures

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.