The stacked-X DNA Holliday junction and protein recognition

The crystal structure of the four-stranded DNA Holliday junction has now been determined in the presence and absence of junction binding proteins, with the extended open-X form of the junction seen in all protein complexes, but the more compact stacked-X structure observed in free DNA. The structures of the stacked-X junction were crystallized because of an unexpected sequence dependence on the stability of this structure. Inverted repeat sequences that contain the general motif NCC or ANC favor formation of stacked-X junctions, with the junction cross-over occurring between the first two positions of the trinucleotides. This review focuses on the sequence dependent structure of the stacked-X junction and how it may play a role in structural recognition by a class of dimeric junction resolving enzymes that themselves show no direct sequence recognition.     

The stability and characteristics of a DNA Holliday junction switch

A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.     

DNA repair defects ascribed to pby1 are caused by disruption of Holliday junction resolvase Mus81-Mms4

PBY1 continues to be linked with DNA repair through functional genomics studies in yeast. Using the yeast knockout (YKO) strain collection, high-throughput genetic interaction screens have identified a large set of negative interactions between PBY1 and genes involved in genome stability. In drug sensitivity screens, the YKO collection pby1Δ strain exhibits a sensitivity profile typical for genes involved in DNA replication and repair. We show that these findings are not related to loss of Pby1. On the basis of genetic interaction profile similarity, we pinpoint disruption of Holliday junction resolvase Mus81-Mms4 as the mutation responsible for DNA repair phenotypes currently ascribed to pby1. The finding that Pby1 is not a DNA repair factor reconciles discrepancies in the data available for PBY1, and indirectly supports a role for Pby1 in mRNA metabolism. Data that has been collected using the YKO collection pby1Δ strain confirms and expands the chemical-genetic interactome of MUS81-MMS4.     

The stalk region of the RecU resolvase is essential for Holliday junction recognition and distortion

The Bacillus subtilis RecU protein has two activities: to recognize, distort, and cleave four-stranded recombination intermediates and to modulate RecA activities. The RecU structure shows a mushroom-like appearance, with a cap and a stalk region. The RuvB interaction and the catalytic residues are located in the cap region of dimeric RecU. We report here that the stalk region is essential not only for RecA modulation but also for Holliday junction (HJ) recognition. Two recU mutants, which map in the stalk region, were isolated and characterized. In vivo, a RecU variant with a Phe81-to-Ala substitution (F81A) was as sensitive to DNA-damaging agents as a null recU strain, and a similar substitution at tyrosine 80 (Y80A) showed an intermediate phenotype. RecUY80A and RecUF81A poorly recognize and distort HJs. RecUY80A cleaves HJs with low efficiency, and RuvB modulates cleavage. At high concentrations, RecUF81A binds to HJs but fails to cleave them. Unlike wild-type RecU, RecUY80A and RecUF81A do not inhibit RecA dATPase and strand-exchange activities. The RecU stalk region is involved in RecA interaction, but once an HJ is bound, RecU fails to modulate RecA activities. Our biochemical study provides a mechanistic basis for the connections between these two mutually exclusive stages (i.e., RecA modulation and HJ resolution) of the recombination reaction.     

Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance.

The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the alpha-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the truncated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif [Ceschini et al. (2001) EMBO J. 20, 6601-6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Deltaydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full-length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA 'phenotype'. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe.     

Cdc14 targets the Holliday junction resolvase Yen1 to the nucleus in early anaphase

The only canonical Holliday junction (HJ) resolvase identified in eukaryotes thus far is Yen1/GEN1. Nevertheless, Yen1/GEN1 appears to have a minor role in HJ resolution, and, instead, other structure-specific endonucleases (SSE) that recognize branched DNA play the leading roles, Mus81-Mms4/EME1 being the most important in budding yeast. Interestingly, cells tightly regulate the activity of each HJ resolvase during the yeast cell cycle. Thus, Mus81-Mms4 is activated in G₂/M, while Yen1 gets activated shortly afterwards. Nevertheless, cytological studies have shown that Yen1 is sequestered out of the nucleus when cyclin-dependent kinase activity is high, i.e., all of the cell cycle but G₁. We here show that the mitotic master phosphatase Cdc14 targets Yen1 to the nucleus in early anaphase through the FEAR network. We will further show that this FEAR-mediated Cdc14-driven event is sufficient to back-up Mus81-Mms4 in removing branched DNA structures, which are especially found in the long chromosome arms upon replication stress. Finally, we found that MEN-driven Cdc14 re-activation in late anaphase is essential to keep Yen1 in the nucleus until the next G₁. Our results highlight the essential role that early-activated Cdc14, i.e., through the FEAR network, has in removing all kind of non-proteinaceous linkages that preclude faithful sister chromatid segregation in anaphase. In addition, our results support the general idea of Yen1 acting as a last resource endonuclease to deal with any remaining HJ that might compromise genetic stability during chromosome segregation.     

The RecQ gene family in plants

RecQ helicases are conserved throughout all kingdoms of life regarding their overall structure and function. They are 3'-5' DNA helicases resolving different recombinogenic DNA structures. The RecQ helicases are key factors in a number of DNA repair and recombination pathways involved in the maintenance of genome integrity. In eukaryotes the number of RecQ genes and the structure of RecQ proteins vary strongly between organisms. Therefore, they have been named RecQ-like genes. Knockouts of several RecQ-like genes cause severe diseases in animals or harmful cellular phenotypes in yeast. Until now the largest number of RecQ-like genes per organism has been found in plants. Arabidopsis and rice possess seven different RecQ-like genes each. In the almost completely sequenced genome of the moss Physcomitrella patens at least five RecQ-like genes are present. One of the major present and future research aims is to define putative plant-specific functions and to assign their roles in DNA repair and recombination pathways in relation to RecQ genes from other eukaryotes. Regarding their intron positions, the structures of six RecQ-like genes of dicots and monocots are virtually identical indicating a conservation over a time scale of 150 million years. In contrast to other eukaryotes one gene (RecQsim) exists exclusively in plants. It possesses an interrupted helicase domain but nevertheless seems to have maintained the RecQ function. Owing to a recent gene duplication besides the AtRecQl4A gene an additional RecQ-like gene (AtRecQl4B) exists in the Brassicaceae only. Genetic studies indicate that a AtRecQl4A knockout results in sensitivity to mutagens as well as an hyper-recombination phenotype. Since AtRecQl4B was still present, both genes must have non-redundant roles. Analysis of plant RecQ-like genes will not only increase the knowledge on DNA repair and recombination, but also on the evolution and radiation of protein families.     

The human Holliday junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant

A key step in meiotic recombination involves the nucleolytic resolution of Holliday junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Δ strains results in Holliday junction resolution and crossover formation during meiosis.     

Holliday junction resolvase in Schizosaccharomyces pombe has identical endonuclease activity to the CCE1 homologue YDC2.

A novel Holliday junction resolving activity has been identified in fractionated cell extracts of the fission yeast Schizosaccharomyces pombe . The enzyme catalyses endonucleolytic cleavage of Holliday junction-containing chi DNA and synthetic four-way DNA junctions. The activity cuts with high specificity a synthetic four-way junction containing a 12 bp core of homologous sequences but has no activity on another four-way junction (with a fixed crossover point), a three-way junction, linear duplex DNA or duplex DNA containing six mismatched nucleotides in the centre. The major cleavage sites map as single nicks in the vicinity of the crossover point, 3' of a thymidine residue. These data indicate that the activity has a strong DNA structure selectivity as well as a limited sequence preference; features similar to the Holliday junction resolving enzymes RuvC of Escherichia coli and the mitochondrial CCE1 (cruciform-cuttingenzyme 1) of Saccharomyces cerevisiae. A putative homologue of CCE1 in S.pombe (YDC2_SCHPO) has been identified through a search of the sequence database. The open reading frame of this gene has been cloned and the encoded protein, YDC2, expressed in E.coli . The purified recombinant YDC2 exhibits Holliday junction resolvase activity and is, therefore, a functional S.pombe homologue of CCE1. The resolvase YDC2 shows the same substrate specificity and produces identical cleavage sites as the activity obtained from S. pombe cells. Both YDC2 and the cellular activity cleave Holliday junctions in both orientations to give nicks that can be ligated in vitro. The partially purified Holliday junction resolving enzyme in fission yeast is biochemically indistinguishable from recombinant YDC2 and appears to be the same protein.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Purification and properties of the RuvA and RuvB proteins of Escherichia coli

Summary The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M_r ca. 100000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and -mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M_r ca. 85000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.     

Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N⁶‐ethenoadenine (εA) is influenced by a minor groove‐interrogating residue more than the composition of the nucleobase‐binding pocket. Exchanging this residue between Mag proteins swapped their εA activities, providing evidence that residues outside the extrahelical base‐binding pocket have a role in identification of a particular modification in addition to sensing damage.     

Molecular dynamics simulation of hydrated d(CGGGTACCCG)4 as a four-way DNA Holliday junction and comparison with the crystallographic structure

Molecular dynamics (MD) simulation of decamer sequence (CGGGTACCCG)₄ as a four-way Holliday junction is reported here for 15.0 ns at three different temperatures 100, 200 and 300 K, respectively, using AMBER force field. Particle mesh Ewald method has been utilised to deal long-range interaction potentials. After MD simulation, various parameters of the junction model including backbone and helical parameters have been worked out and the dynamical pathway is discussed. Structural analysis and geometrical calculations were carried out through X3DNA. The computational results obtained are compared with the previously reported crystallographic outcomes. The width and depth of the major and minor grooves of the duplex of the four arms of the DNA junction have been calculated. The variations in the C1′–C1′ distances between the two complementary strands are discussed in detail. A close observation of the results reveals that the conformation of the average simulated structure at low temperature is of ‘B’ form and the structural integrity of the DNA junction having a twofold sequence symmetry is temperature dependent. It also seems that besides the other parameters (i.e. presence of ions, solvents, etc.), temperature may be playing a key role in preserving the structural integrity of the DNA junction.     

Escherichia coli RuvA and RuvB proteins involved in recombination repair: physical properties and interactions with DNA

Summary Escherichia coli RuvA and RuvB proteins are encoded by an SOS-regulated operon, which is involved in DNA repair and recombination. RuvB has weak ATPase activity, which is enhanced by the addition of RuvA and DNA, and RuvA and RuvB in the presence of ATP promote branch migration at Holliday junctions. In this work, the physical states of RuvA and RuvB and their interactions with DNA were studied by sedimentation analysis and gel filtration chromatography. RuvA formed a stable tetramer in solution, which resisted dissociation by SDS at room temperature. RuvB formed a dimer in solution. When RuvA and RuvB were mixed, an oligomer complex was formed consisting of a tetrameric form of RuvA and a dimeric form of RuvB, and this complex bound to DNA. The maximal enhancement of the RuvB ATPase activity by RuvA was achieved at this stoichiometry in the presence of excess DNA.     

Repair of deaminated base damage by Schizosaccharomyces pombe thymine DNA glycosylase

Thymine DNA glycosylases (TDG) in eukaryotic organisms are known for their double-stranded glycosylase activity on guanine/uracil (G/U) base pairs. Schizosaccharomyces pombe (Spo) TDG is a member of the MUG/TDG family that belongs to a uracil DNA glycosylase superfamily. This work investigates the DNA repair activity of Spo TDG on all four deaminated bases: xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine, and uracil from cytosine. Unexpectedly, Spo TDG exhibits glycosylase activity on all deaminated bases in both double-stranded and single-stranded DNA in the descending order of X > I > U  O. In comparison, human TDG only excises deaminated bases from G/U and, to a much lower extent, A/U and G/I base pairs. Amino acid substitutions in motifs 1 and 2 of Spo TDG show a significant impact on deaminated base repair activity. The overall mutational effects are characterized by a loss of glycosylase activity on oxanine in all five mutants. L157I in motif 1 and G288M in motif 2 retain xanthine DNA glycosylase (XDG) activity but reduce excision of hypoxanthine and uracil, in particular in C/I, single-stranded hypoxanthine (ss-I), A/U, and single-stranded uracil (ss-U). A proline substitution at I289 in motif 2 causes a significant reduction in XDG activity and a loss of activity on C/I, ss-I, A/U, C/U, G/U, and ss-U. S291G only retains reduced activity on T/I and G/I base pairs. S163A can still excise hypoxanthine and uracil in mismatched base pairs but loses XDG activity, making it the closest mutant, functionally, to human TDG. The relationship among amino acid substitutions, binding affinity and base recognition is discussed.     

Conformational and hydration effects of site-selective sodium,calcium and strontium ion binding to the DNA Holliday junction structure d(TCGGTACCGA)(4)

The role of metal ions in determining the solution conformation of the Holliday junction is well established, but to date the picture of metal ion binding from structural studies of the four-way DNA junction is very incomplete. Here we present two refined structures of the Holliday junction formed by the sequence d(TCGGTACCGA) in the presence of Na(+) and Ca(2+), and separately with Sr(2+) to resolutions of 1.85A and 1.65A, respectively. This sequence includes the ACC core found to promote spontaneous junction formation, but its structure has not previously been reported. Almost complete hydration spheres can be defined for each metal cation. The Na(+) sites, the most convincing observation of such sites in junctions to date, are one on either face of the junction crossover region, and stabilise the ordered hydration inside the junction arms. The four Ca(2+) sites in the same structure are at the CG/CG steps in the minor groove. The Sr(2+) ions occupy the TC/AG, GG/CC, and TA/TA sites in the minor groove, giving ten positions forming two spines of ions, spiralling through the minor grooves within each arm of the stacked-X structure. The two structures were solved in the two different C2 lattices previously observed, with the Sr(2+) derivative crystallising in the more highly symmetrical form with two-fold symmetry at its centre. Both structures show an opening of the minor groove face of the junction of 8.4 degrees in the Ca(2+) and Na(+) containing structure, and 13.4 degrees in the Sr(2+) containing structure. The crossover angles at the junction are 39.3 degrees and 43.3 degrees, respectively. In addition to this, a relative shift in the base pair stack alignment of the arms of 2.3A is observed for the Sr(2+) containing structure only. Overall these results provide an insight into the so-far elusive stabilising ion structure for the DNA Holliday junction.     

The role of the SAP motif in promoting Holliday junction binding and resolution by SpCCE1

Holliday junctions are four-way branched DNA structures that are formed during recombination and by replication fork regression. Their processing depends on helicases that catalyze junction branch migration, and endonucleases that resolve the junction into nicked linear DNAs. Here we have investigated the role of a DNA binding motif called SAP in binding and resolving Holliday junctions by the fission yeast mitochondrial resolvase SpCCE1. Mutation or partial/complete deletion of the SAP motif dramatically impairs the ability of SpCCE1 to resolve Holliday junctions in a heterologous in vivo system. These mutant proteins retain the ability to recognize the junction structure and to distort it upon binding. However, once formed the mutant protein-junction complexes are relatively unstable and dissociate much faster than wild-type complexes. We show that binding stability is necessary for efficient junction resolution, and that this may be due in part to a requirement for maintaining the junction in an open conformation so that it can branch migrate to cleavable sites.     

Electrostatic interactions and the folding of the four-way DNA junction: analysis by selective methyl phosphonate substitution

The structure and dynamics of the four-way (Holliday) junction are strongly dependent on the presence of metal ions. In this study, the importance of phosphate charge in and around the point of strand exchange has been explored by selective replacement with electrically neutral methyl phosphonate groups, guided by crystal structures of the junction in the folded, stacked X conformation. Junction conformation has been analysed by comparative gel electrophoresis and fluorescence resonance energy transfer (FRET). Three of sets of phosphate groups on the exchanging strands have been analysed; those at the point of strand exchange and those to their 3' and 5' sides. The exchanging and 3' phosphate groups form a box of negatively charged groups on the minor groove face of the junction, while the 5' phosphate groups face each other on the major groove side, with their proR oxygen atoms directed at one another. The largest effects are observed on substitution of the exchanging phosphate groups; replacement of both groups leads to the loss of the requirement for addition of metal ions to allow junction folding. When the equivalent phosphate groups on the continuous strands were substituted, a proportion of the junction folded into the alternative conformer so as to bring these phosphate groups onto the exchanging strands. These species did not interconvert, and thus this is likely to result from the alternative diasteromeric forms of the methyl phosphonate group. This shows that some of the conformational effects result from more than purely electrostatic interactions. Smaller but significant effects were observed on substitution of the flanking phosphate groups. All methyl phosphonate substitutions at these positions allowed folding to proceed at a reduced concentration of magnesium ions, with double substitutions more effective than single substitutions. Substitution of 5' phosphates resulted in a greater degree of folding at a given ionic concentration compared to the corresponding 3' phosphate substitutions. These results show that the phosphate groups at the point of strand exchange exert the largest electrostatic effect on junction folding, but a number of phosphate groups in the vicinity of the exchange region contribute to the overall effects.     

AtRECQ2, a RecQ helicase homologue from Arabidopsis thaliana, is able to disrupt various recombinogenic DNA structures in vitro

RecQ helicases play an important role in the maintenance of genomic stability in pro- and eukaryotes. This is highlighted by the human genetic diseases Werner, Bloom's and Rothmund–Thomson syndrome, caused by respective mutations in three of the five human RECQ genes. The highest numbers of RECQ homologous genes are found in plants, e.g. seven in Arabidopsis thaliana . However, only limited information is available on the functions of plant RecQ helicases, and no biochemical characterization has been performed. Here, we demonstrate that AtRECQ2 is a (d)NTP-dependent 3'→5' DNA helicase. We further characterized its basal properties and its action on various partial DNA duplexes. Importantly, we demonstrate that AtRECQ2 is able to disrupt recombinogenic structures: by disrupting various D-loop structures, AtRECQ2 may prevent non-productive recombination events on the one hand, and may channel repair processes into non-recombinogenic pathways on the other hand, thus facilitating genomic stability. We show that a synthetic partially mobile Holliday junction is processed towards splayed-arm products, possibly indicating a branch migration function for AtRECQ2. The biochemical properties defined in this work support the hypothesis that AtRECQ2 might be functionally orthologous to the helicase part of the human RecQ homologue HsWRN.     

Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex

During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.