Two Holliday junction resolving enzymes in Sulfolobus solfataricus

Holliday junction resolving enzymes bind specifically to four-way DNA junctions created by the process of homologous recombination, cleaving them to yield recombinant duplex DNA products. Homologous recombination is known to occur in the third domain of life, the archaea, and may constitute a simplified model for the corresponding eucaryal pathway, but has not been well characterised. Identification of a gene encoding an archaeal Holliday junction resolving enzyme, Hjc, has recently been reported in the euryarchaea, and an activity has been observed in the hyperthermophilic crenarchaeote Sulfolobus solfataricus. Here we report the identification, heterologous expression and characterisation of the Hjc protein from Sulfolobus. We demonstrate that Sulfolobus has two distinct junction resolving enzymes, Hjc and Hje, with differing substrate specificities.     

A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc

Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3' to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.     

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.     

Multiple Holliday junction resolving enzyme activities in the Crenarchaeota and Euryarchaeota

Holliday junction resolving enzymes are required by all life forms that catalyse homologous recombination, including all cellular organisms and many bacterial and eukaryotic viruses. Here we report the identification of three distinct Holliday junction resolving enzyme activities present in two highly divergent archaeal species. Both Sulfolobus and Pyrococcus share the Hjc activity, and in addition possess unique secondary activities (Hje and Hjr). We propose by analogy with the two other domains of life that the latter enzymes are viral in origin, suggesting the widespread existence of archaeal viruses that rely on homologous recombination as part of their life cycle.     

Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje

Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns--they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.     

Dissection of the functional domains of an archaeal Holliday junction helicase

Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHjm) and its truncated derivatives, and characterization of the StoHjm proteins revealed that the N-terminal module (residues 1-431) alone was capable of ATP hydrolysis and DNA binding, while the C-terminal one (residues 415-704) was responsible for regulating the helicase activity. The region involved in StoHjm-StoHjc (Hjc from S. tokodaii) interaction was identified as part of domain II, domain III (Winged Helix motif), and domain IV (residues 366-645) for StoHjm. We present evidence supporting that StoHjc regulates the helicase activity of StoHjm by inducing conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting that Hjm/Hjc mediated resolution of stalled replication forks is of crucial importance in archaea. A tentative pathway with which Hjm/Hjc interaction could have occurred at stalled replication forks is discussed.     

Genetic analysis of an archaeal Holliday junction resolvase in Escherichia coli.

The study of genes and proteins in heterologous model systems provides a powerful approach to the analysis of common processes in biology. Here, we show how the bacterium Escherichia coli can be exploited to analyse genetically and biochemically the activity and function of a Holliday junction resolving enzyme from an archaeal species. We have purified and characterised a member of the newly discovered Holliday junction cleaving (Hjc) family of resolvases from the moderately thermophilic archaeon Methanobacterium thermoautotrophicum and demonstrate that it promotes DNA repair in resolvase-deficient ruv mutants of E. coli. The data presented provide the first direct evidence that such archaeal enzymes can promote DNA repair in vivo, and support the view that formation and resolution of Holliday junctions are key to the interplay between DNA replication, recombination and repair in all organisms. We also show that Hjc promotes DNA repair in E. coli in a manner that requires the presence of the RecG branch migration protein. These results support models in which RecG acts at a replication fork stalled at a lesion in the DNA, catalysing fork regression and forming a Holliday junction that can then be acted upon by Hjc.     

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.     

Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I

We have solved the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 A resolution using the multiwavelength anomalous dispersion (MAD) technique. Endonuclease I exhibits strong structural specificity for four-way DNA junctions. The structure shows that it forms a symmetric homodimer arranged in two well-separated domains. Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site. While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases. T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes.     

Holliday junction resolution is modulated by archaeal chromatin components in vitro.

The Holliday junction-resolving enzyme Hjc is conserved in the archaea and probably plays a role analogous to that of Escherichia coli RuvC in the pathway of homologous recombination. Hjc specifically recognizes four-way DNA junctions, cleaving them without sequence preference to generate recombinant DNA duplex products. Hjc imposes an X-shaped global conformation on the bound DNA junction and distorts base stacking around the point of cleavage, three nucleotides 3' of the junction center. We show that Hjc is autoinhibitory under single turnover assay conditions and that this can be relieved by the addition of either competitor duplex DNA or the architectural double-stranded DNA-binding protein Sso7d (i.e. by approximating in vivo conditions more closely). Using a combination of isothermal titration calorimetry and fluorescent resonance energy transfer, we demonstrate that multiple Hjc dimers can bind to each synthetic four-way junction and provide evidence for significant distortion of the junction structure at high protein:DNA ratios. Analysis of crystal packing interactions in the crystal structure of Hjc suggests a molecular basis for this autoinhibition. The wider implications of these findings for the quantitative study of DNA-protein interactions is discussed.     

A novel virus family, the Rudiviridae: Structure, virus-host interactions and genome variability of the sulfolobus viruses SIRV1 and SIRV2.

The unenveloped, stiff-rod-shaped, linear double-stranded DNA viruses SIRV1 and SIRV2 from Icelandic Sulfolobus isolates form a novel virus family, the Rudiviridae. The sizes of the genomes are 32. 3 kbp for SIRV1 and 35.8 kbp for SIRV2. The virions consist of a tube-like superhelix formed by the DNA and a single basic 15.8-kD DNA-binding protein. The tube carries a plug and three tail fibers at each end. One turn of the DNA-protein superhelix measures 4.3 nm and comprises 16.5 turns of B DNA. The linear DNA molecules appear to have covalently closed hairpin ends. The viruses are not lytic and are present in their original hosts in carrier states. Both viruses are quite stable in these carrier states. In several laboratory hosts SIRV2 was invariant, but SIRV1 formed many different variants that completely replaced the wild-type virus. Some of these variants were still variable, whereas others were stable. Up to 10% nucleotide substitution was found between corresponding genome fragments of three variants. Some variants showed deletions. Wild-type SIRV1, but not SIRV2, induces an SOS-like response in Sulfolobus. We propose that wild-type SIRV1 is unable to propagate in some hosts but surmounts this host range barrier by inducing a host response effecting extensive variation of the viral genome.     

Dissection of the regional roles of the archaeal Holliday junction resolvase Hjc by structural and mutational analyses.

Hjc is an archaeal DNA endonuclease, which resolves the Holliday junction in the presence of divalent metals. Combined with mutational analyses, the x-ray structure of the Pyrococcus furiosus Hjc crystal grown in the presence of ammonium sulfate revealed a positively charged interface, rich in conserved basic residues, and the catalytic center (Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y., and Morikawa, K. (2001) Structure 9, 197-T204). This structural study also suggested that the N-terminal segment and some loops of Hjc play crucial roles in the cleavage of DNA. However, a structural view of the interaction between these regions and DNA remains elusive. To clarify the regional roles of Hjc in the recognition of the Holliday junction, further structural and biochemical analyses were carried out. A new crystal form of Hjc was obtained from a polyethylene glycol solution in the absence of ammonium sulfate, and its structure has been determined at 2.16-A resolution. A comparison of the two crystal structures has revealed that the N-terminal segment undergoes a serious conformational change. The site-directed mutagenesis of the sulfate-binding site within the segment caused a dramatic decrease in the junction binding, but the mutant was still capable of cleaving DNA with a 20-fold lower efficiency. The kinetic analysis of Hjc-Holliday junction interaction indicated that mutations in the N-terminal segment greatly increased the dissociation rate constants of the Hjc-Holliday junction complex, explaining the decreased stability of the complex. This segment is also responsible for the disruption of base pairs near the junction center, through specific interactions with them. Taken together, these results imply that, in addition to the secondary effects of two basic loops, the flexible N-terminal segment plays predominant roles in the recognition of DNA conformation near the crossover and in correct positioning of the cleavage site to the catalytic center of the Hjc resolvase.     

Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1

Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange. In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques. Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium. The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold. This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments [Overmars and Altona (1997) J. Mol. Biol. 273, 519-524; Panyutin et al. (1995) EMBO J. 14, 1819-1826]. The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.     

Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.     

Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1

Yen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or the increase in the first incision rate by assembly of a dimer on a HJ, despite minor differences. However, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5′-3′ exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfils the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.     

Definitions and analysis of DNA Holliday junction geometry

A number of single-crystal structures have now been solved of the four-stranded antiparallel stacked-X form of the Holliday junction. These structures demonstrate how base sequence, substituents, and drug and ion interactions affect the general conformation of this recombination intermediate. The geometry of junctions had previously been described in terms of a specific set of parameters that include: (i) the angle relating the ends of DNA duplexes arms of the junction (interduplex angle); (ii) the relative rotation of the duplexes about the helix axes of the stacked duplex arms (J_roll); and (iii) the translation of the duplexes along these helix axes (J_slide). Here, we present a consistent set of definitions and methods to accurately calculate each of these parameters based on the helical features of the stacked duplex arms in the single-crystal structures of the stacked-X junction, and demonstrate how each of these parameters contributes to an overall conformational feature of the structure. We show that the values for these parameters derived from global rather than local helical axes through the stacked bases of the duplex arms are the most representative of the stacked-X junction conformation. In addition, a very specific parameter (J_twist) is introduced which relates the relative orientation of the stacked duplex arms across the junction which, unlike the interduplex angle, is length independent. The results from this study provide a general means to relate the geometric features seen in the crystal structures to those determined in solution.     

Recombination: Holliday junction resolution and crossover formation

The heterodimeric nuclease Mus81-Eme1 has been proposed to be a Holliday junction resolvase and has now been found to be responsible for nearly all meiotic crossovers in fission yeast. The intriguing substrate preference of this enzyme for nicked Holliday junctions opens the possibility that crossover formation may not always involve double Holliday junctions.     

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.