Interaction of the resolving enzyme YDC2 with the four-way DNA junction

Holliday junctions (four-way DNA junctions), formed during homologous recombination, are bound and resolved by junction-specific endonucleases to yield recombinant duplex DNA products. The junction-resolving enzymes are a structurally diverse class of proteins that nevertheless have many properties in common; in particular a high structure specificity for binding and metal-dependent, (frequently) sequence-specific cleavage activity. In Saccharomyces cerevisiae, the enzyme CCE1 is necessary for the resolution of recombining mitochondrial genomes, and in Schizosaccharomyces pombe the homologous protein YDC2 is thought to have a similar function. We have generated an inactive mutant of YDC2, D226N, that retains structure-specific junction binding and have analysed the interaction of this protein with the four-way DNA junction. YDC2 binds the four-way junction in two specific complexes (I and II), unfolding the stacked X-structure into a conformation where the arms extend to the four corners of a square. This structure is reminiscent of that of the free junction in the absence of metal ions and of the structures imposed on the Holliday junction by CCE1 and RuvA. DNase I probing reveals footprints specific for complexes I and II which extend from the junction centre on all four arms. No protection is observed with the small, hydrophobic probe DMS.     

Dissection of the sequence specificity of the Holliday junction endonuclease CCE1

CCE1 is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomycescerevisiae. The junction-resolving enzymes are a diverse class, widely distributed in nature from viruses to higher eukaryotes. In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent. We have undertaken a systematic study of the sequence specificity of CCE1, using a single-turnover kinetic assay and a panel of synthetic four-way DNA junction substrates. A tetranucleotide consensus cleavage sequence 5'-ACT downward arrowA has been identified, with specificity residing mainly at the central CT dinucleotide. Equilibrium constants for CCE1 binding to four-way junctions are unaffected by sequence variations, suggesting that substrate discrimination occurs predominantly in the transition state complex. CCE1 cuts most efficiently at the junction center, but can also cleave the DNA backbone at positions one nucleotide 3' or 5' of the point of strand exchange, suggesting a significant degree of conformational flexibility in the CCE1:junction complex. Introduction of base analogues at single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail. In particular, the N7 moiety of the guanine base-pairing with the cytosine of the consensus sequence appears to be crucial for catalysis. The functional significance of sequence specificity in junction-resolving enzymes is discussed.     

Analysis of substrate specificity of the RuvC holliday junction resolvase with synthetic Holliday junctions

The Escherichia coli RuvC protein endonucleolytically resolves Holliday junctions, which are formed as intermediates during genetic recombination and recombination repair. Previous studies using model Holliday junctions suggested that a certain size of central core of homology and a specific sequence in the junction were required for efficient cleavage by RuvC, although not for binding. To determine the minimum length of sequence homology required for RuvC cleavage, we made a series of synthetic Holliday junctions with various lengths of homologous sequence in the core region. It was demonstrated that a monomobile junction possessing only 2 base pairs of the homology core was efficiently cleaved by RuvC. To study the sequence specificity for cleavage, we made 16 bimobile junctions, which differed only in the homologous core sequence. Among them, 6 junctions were efficiently cleaved. Cleavage occurred by introduction of nicks symmetrically at the 3'-side of thymine in all cases. However, the nucleotide bases at the 3'-side of the thymines were not always identical between the two strands nicked. These results suggest that RuvC recognizes mainly topological symmetry of the Holliday junction but not the sequence symmetry per se, that the thymine residue at the cleavage site plays an important role for RuvC-mediated resolution, and that a long homologous core sequence is not essential for cleavage.     

Substrate specificity of the Escherichia coli RuvC protein. Resolution of three- and four-stranded recombination intermediates

The specificity of the Escherichia coli RuvC Holliday junction resolvase has been investigated in vitro. RuvC protein cleaves synthetic DNA substrates that model three- or four-stranded recombination intermediates but fails to act upon Y junctions, G/A mismatches, heterologous loop structures, or two-stranded branched junctions. RuvC therefore differs from endonuclease VII of bacteriophage T4 which exhibits broad range specificity. Using related three- and four-stranded synthetic DNA junctions, we show that RuvC cleaves both junctions at the same DNA sequence and requires a region of homology at the junction point. The action of RuvC on three- and four-stranded recombination intermediates made by RecA was also investigated. We found that RuvC fails to resolve three-stranded intermediates in the presence of RecA, although four-stranded intermediates are resolved under the same conditions. However, both three- and four-stranded intermediates are substrates for the nuclease after removal of RecA. We interpret these differences in terms of the contiguity of the RecA nucleoprotein filament which may, under certain conditions, limit access to the Holliday junction resolvase.     

Formation of RuvABC-Holliday junction complexes in vitro

In Escherichia coli, the RuvA, RuvB and RuvC proteins are required for the late stages of homologous recombination and DNA repair. RuvA and RuvB form a complex that interacts with Holliday junctions--crossed DNA structures that are recombination intermediates--and promotes branch migration; RuvC is a junction-specific endonuclease that resolves Holliday junctions and completes the recombination process. Because genetic and biochemical experiments suggest that the processes of branch migration and resolution are linked, coimmunoprecipitation experiments were carried out to determine whether the three Ruv proteins interact to form a functional complex (RuvABC). Using a synthetic Holliday junction, a multisubunit complex containing the junction and RuvA, RuvB and RuvC was detected. In the absence of RuvB, RuvAC-junction complexes were observed. Complex formation was not facilitated by duplex DNA. The identification of a RuvABC-junction complex provides direct evidence that the RuvABC proteins interact at the Holliday junction.     

Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion

E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using small synthetic junctions, we show that junction recognition is structure-specific and occurs in the absence of metal cofactors. In the presence of Mg2+, Holliday junctions are resolved by the introduction of symmetrically related nicks at the 3' side of thymine residues. The nicked duplex products are repaired by the action of DNA ligase. Within the RuvC-Holliday junction complex, the DNA is distorted such that 2 of the 4 strands become hypersensitive to hydroxyl radical attack. The ionic requirements of binding, hydroxyl radical sensitivity, and strand cleavage indicate three distinct steps in the mechanism of RuvC-mediated Holliday junction resolution: structure-specific recognition, DNA distortion, and sequence-dependent cleavage.     

The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae

CCE1 is a DNA junction-resolving enzyme involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. The CCE1 gene was cloned by PCR, and the expressed protein purified to homogeneity. CCE1 was found to bind to four-way DNA junctions, with a strong structural selectivity. The enzyme binds DNA junctions as a dimer, with slow subunit exchange occurring in free solution. While CCE1 binds equally to synthetic four-way DNA junctions of any sequence, it exhibits pronounced sequence-selectivity in cleavage. Both fixed junctions and those capable of branch migration can be cleaved, with a preference for cleavage at the sequence 5'CT/. Cleavage of junctions tethered to adopt specific stacking isomers demonstrated that the target sequences are cleaved fivefold faster when located on a continuous strand compared to an exchanging strand.     

Recognition and manipulation of branched DNA by the RusA Holliday junction resolvase of Escherichia coli

Homologous recombination is a fundamental cellular process that shapes and reshapes the genomes of all organisms and promotes repair of damaged DNA. A key step in this process is the resolution of Holliday junctions formed by homologous DNA pairing and strand exchange. In Escherichia coli , a Holliday junction is processed into recombinant products by the concerted activities of the RuvA and RuvB proteins, which together drive branch migration, and RuvC endonuclease, which resolves the structure. In the absence of RuvABC, recombination can be promoted by increasing the expression of the RusA endonuclease, a Holliday junction resolvase encoded by a cryptic prophage gene. Here, we describe the DNA binding properties of RusA. We found that RusA was highly selective for branched molecules and formed complexes with these structures even in the presence of a large excess of linear duplex DNA. However, it does bind weakly to linear duplex DNA. Under conditions where there was no detectable binding to duplex DNA, RusA formed a highly structured complex with a synthetic Holliday junction that was remarkably stable and insensitive to divalent metal ions. The duplex arms were found to adopt a specific alignment within this complex that approximated to a tetrahedral conformation of the junction.     

Twin-twin transfusion syndrome: the challenge of etiology-based management decisions

Here we present the crystal structure of the Escherichia coli protein RuvA bound to a key DNA intermediate in recombination, the Holliday junction. The structure, solved by isomorphous replacement and density modification at 6 A resolution, reveals the molecular architecture at the heart of the branch migration and resolution reactions required to process Holliday intermediates into recombinant DNA molecules. It also reveals directly for the first time the structure of the Holliday junction. A single RuvA tetramer is bound to one face of a junction whose four DNA duplex arms are arranged in an open and essentially four-fold symmetric conformation. Protein-DNA contacts are mediated by two copies of a helix-hairpin-helix motif per RuvA subunit that contact the phosphate backbone in a very similar manner. The open structure of the junction stabilized by RuvA binding exposes a DNA surface that could be bound by the RuvC endonuclease to promote resolution.     

Recognition and manipulation of branched DNA structure by junction-resolving enzymes

The junction-resolving enzymes are a class of nucleases that introduce paired cleavages into four-way DNA junctions. They are important in DNA recombination and repair, and are found throughout nature, from eubacteria and their bacteriophages through to higher eukaryotes and their viruses. These enzymes exhibit structure-selective binding to DNA junctions; although cleavage may be more or less sequence-dependent, binding affinity is purely related to the branched structure of the DNA. Binding and cleavage events can be separated for a number of the enzymes by mutagenesis, and mutant proteins that are defective in cleavage while retaining normal junction-selective binding have been isolated. Critical acidic residues have been identified in several resolving enzymes, suggesting a role in the coordination of metal ions that probably deliver the hydrolytic water molecule. The resolving enzymes all bind to junctions in dimeric form, and the subunits introduce independent cleavages within the lifetime of the enzyme-junction complex to ensure resolution of the four-way junction. In addition to recognising the structure of the junction, recent data from four different junction-resolving enzymes indicate that they also manipulate the global structure. In some cases this results in severe distortion of the folded structure of the junction. Understanding the recognition and manipulation of DNA structure by these enzymes is a fascinating challenge in molecular recognition.     

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.     

Identification of three aspartic acid residues essential for catalysis by the RusA holliday junction resolvase

RusA is a Holliday junction resolvase encoded by the cryptic prophage DLP12 of Escherichia coli K-12 that can be activated to promote homologous recombination and DNA repair in resolution-deficient mutants lacking the RuvABC proteins. Database searches with the 120 amino acid residue RusA sequence identified 11 homologues from diverse species, including one from the extreme thermophile Aquifex aeolicus, which suggests that RusA may be of ancient bacterial ancestry. A multiple alignment of these sequences revealed seven conserved or invariant acidic residues in the C-terminal half of the E. coli protein. By making site-directed mutations at these positions and analysing the ability of the mutant proteins to promote DNA repair in vivo and to resolve junctions in vitro, we identified three aspartic acid residues (D70, D72 and D91) that are essential for catalysis and that provide the first insight into the active-site mechanism of junction resolution by RusA. Substitution of any one of these three residues with asparagine reduces resolution activity >80-fold. The mutant proteins retain the ability to bind junction DNA regardless of the DNA sequence or of the mobility of the crossover. They interfere with the function of the RuvABC proteins in vivo, when expressed from a multicopy plasmid, an effect that is reproducible in vitro and that reflects the fact that the RusA proteins have a higher affinity for junction DNA in the presence of Mg2+ than do the RuvA and RuvC proteins. The D70N protein has a greater affinity for junctions in Mg2+ than does the wild-type, which indicates that the negatively charged carboxyl group of the aspartate residue plays a critical role at the active site of RusA. Electrostatic repulsions between D70, D72 and D91 may help to form a classical Mg2+-binding pocket.     

Formation of a single base mismatch impedes spontaneous DNA branch migration

DNA branch migration, a process whereby two homologous DNA duplexes exchange strands, is an essential component of genetic recombination. Models for homologous recombination have invoked spontaneous branch migration as one mechanism for the generation of large regions of heteroduplex DNA. During recombination, two homologous parental duplexes that contain similar, but not identical, sequences are paired and undergo strand exchange. An important issue is whether spontaneous branch migration is capable of traversing sequence heterology such as mismatches, insertions and deletions. We use a model four-strand system to examine the effect of mispaired or unpaired bases on branch migration. The assay consists of annealing two short duplexes having defined sequence heterologies. Following annealing, a Holliday junction is formed that is free to branch migrate. Our results demonstrate that a single base mismatch, insertion or deletion is sufficient to pose a substantial barrier to spontaneous branch migration. In the presence of magnesium, branch migration through such sequence heterologies is almost completely blocked. Others have shown that non-mobile four-way junctions undergo a dramatic shift in conformation in the presence of magnesium. Our data suggest that a similar transition occurs for the mobile Holliday junction. We also discuss how proteins may facilitate branch migration through sequence heterologies in vivo.     

Characterization of the low magnification performance of a Philips CM300-FEG

BACKGROUND: All the ruvA, ruvB and ruvC mutants of Escherichia coli are sensitive to treatments that damage DNA, and are mildly defective in homologous recombination. It has been reported that the ruv mutants form nonseptate, multinuclear filaments after low doses of UV irradiation, dependent on the sfiA gene product. In vitro, the RuvAB complex promotes the branch migration of Holliday junctions, and RuvC resolves the junctions endonucleolytically. RESULTS: After a low UV dose (5 J/m2), both delta ruvAB and delta ruvC mutant cells became filamentous, with their chromosomes aggregated in the central region. This corresponded to an increase in nonmigrating DNA on pulsed field gel electrophoresis of the XbaI digested chromosome. Upon further incubation, they produced a large number of anucleoid cells of normal size. A recA mutation, but not a recB mutation, suppressed these phenotypes of the ruv mutants. The ruv polA12(Ts) double mutants were inviable at the nonpermissive temperature and mimicked the morphological phenotypes of the UV irradiated ruv mutants. CONCLUSION: ruvA, B and C mutations block chromosome partitioning in UV irradiated cells because the abortive homologous recombination covalently links chromosomes together. There is a recBCD independent pathway for the recA dependent formation of recombination intermediates. An Ruv-mediated resolution of recombination intermediates is required for the repair of strand breaks produced in UV irradiated cells and in the polA mutant cells.     

Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase

Binding of a cleavage-incompetent mutant of the Flp recombinase induces a roughly square-planar geometry in synthetic immobile Holliday junctions. The branch points, which are rigidly fixed in these junctions in their free forms, tend to be more flexible in their protein-bound forms. Our results (1) suggest a plausible mechanism for the switching of the recombination complex from the Holliday-forming mode to the Holliday-resolving mode, (2) provide a rationale for previous observations that Flp resolves preformed immobile Holliday structures in the parental or in the recombinant mode in a relatively unbiased manner, and (3) accommodate two modes of DNA cleavage by Flp (transhorizontal or transdiagonal) in Holliday substrates.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

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.     

Expression of alpha3, beta3 and gamma1 GABA(A) receptor subunit messenger RNAs in visual cortex and lateral geniculate nucleus of normal and monocularly deprived monkeys

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium leprae RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RG values for the free and complexed protein in 65% 2H2O, where the DNA component is "invisible", showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (x10(-5)), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1-2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.     

High resolution computed tomography (HRCT) of pulmonary infections in AIDS patients

The Escherichia coli RuvA and RuvB proteins mediate ATP-dependent branch migration of Holliday junctions during homologous genetic recombination. RuvA is a DNA-binding protein with high affinity for Holliday junctions, to which it directs RuvB (a DNA-dependent ATPase). Electron microscopic studies have shown that RuvB forms double hexameric rings on duplex DNA. To determine whether the rings are biologically active, the conditions required for their formation and activity have been analysed. The quaternary structure of RuvB appears to be dependent upon the binding of ATP, magnesium ions, and the presence of RuvA. In the presence of Mg2+ and ATP, RuvB forms hexamers; however, in the presence of Mg2+ alone, dodecamers were observed. Both forms of the protein are stable and have been isolated by gel filtration. Performed dodecamers and, to a lesser extent, hexamers assembled in the absence of DNA lack ATPase activity. Maximal ATPase activity was observed when RuvB assembled directly on DNA in the presence of Mg2+ and ATP. Moreover, under these conditions, a direct interaction between RuvB hexamers and tetramers of RuvA was observed.