MOC1 cleaves Holliday junctions through a cooperative nick and counter-nick mechanism mediated by metal ions.

Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions.

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6.

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Šresolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.

Novel insights into ATP-Stimulated Cleavage of branched DNA and RNA Substrates through Structure-Guided Studies of the Holliday Junction Resolvase RuvX.

Much of our understanding of the homologous recombination (HR) machinery hinges on studies using Escherichia coli as a model organism. Interestingly enough, studies on the HR machinery in different bacterial species casts doubt on the universality of the E. coli paradigm. The human pathogen Mycobacterium tuberculosis encodes two Holliday junction (HJ)-resolvase paralogues, namely RuvC and RuvX; however, insights into their structural features and functional relevance is still limited. Here, we report on structure-guided functional studies of the M. tuberculosis RuvX HJ resolvase (MtRuvX). The crystalline MtRuvX is a dimer in the asymmetric unit, and each monomer has a RNAse H fold vis-à-vis RuvC-like nucleases. Interestingly, MtRuvX also contains some unique features, including the residues essential for ATP binding/coordination of Mg2+ ions. Indeed, MtRuvX exhibited an intrinsic, robust ATPase activity, which was further accentuated by DNA cofactors. Structure-guided substitutions of single residues at the ATP binding/Mg2+coordination sites while markedly attenuating the ATPase activity completely abrogated HJ cleavage, indicating an unanticipated relationship between ATP hydrolysis and DNA cleavage. However, the affinity of ATPase-deficient mutants for the HJ was not impaired. Contrary to RuvC, MtRuvX exhibits relaxed substrate specificity, cleaving a variety of branched DNA/RNA substrates. Notably, ATP hydrolysis plays a regulatory role, rendering MtRuvX from a canonical HJ resolvase to a DNA/RNA non-sequence specific endonuclease, indicating a link between HJ resolvase and nucleic acid metabolism. These findings provide novel insights into the structure and dual-functional activities of MtRuvX, and suggest that it may play an important role in DNA/RNA metabolism.

Biochemical and structural characterization of the Holliday junction resolvase RuvC from Pseudomonas aeruginosa.

The Holliday junction, a four-way DNA structure, is an important intermediate of homologous recombination. Proper Holliday junction resolution is critical to complete the recombination process. In most bacterial cells, the Holliday junction cleavage is mainly performed by a specific endonuclease RuvC. Here, we describe the biochemical properties and the crystal structure of RuvC from an opportunistic pathogen, Pseudomonas aeruginosa (PaRuvC). PaRuvC specifically binds to the Holliday junction DNA and preferentially cleaves it at the consensus 5'-TTC-3'. PaRuvC uses Mg2+ as the preferred divalent metal cofactor for Holliday junction cleavage and its optimum pH is 8.0-9.0. Elevated temperatures (37-60 °C) boost the catalytic activity, but temperatures higher than 53 °C reduce the protein stability. The crystal structure of PaRuvC determined at 2.4 Å and mutagenesis analysis reveal key residues involved in the dimer formation, substrate binding and catalysis. Our results are expected to provide useful information to combat antibiotic resistance of Pseudomonas aeruginosa by targeting its homologous recombination system.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

Structural basis of sequence-specific Holliday junction cleavage by MOC1.

The Holliday junction (HJ) is a key intermediate during homologous recombination and DNA double-strand break repair. Timely HJ resolution by resolvases is critical for maintaining genome stability. The mechanisms underlying sequence-specific substrate recognition and cleavage by resolvases remain elusive. The monokaryotic chloroplast 1 protein (MOC1) specifically cleaves four-way DNA junctions in a sequence-specific manner. Here, we report the crystal structures of MOC1 from Zea mays, alone or bound to HJ DNA. MOC1 uses a unique ß-hairpin to embrace the DNA junction. A base-recognition motif specifically interacts with the junction center, inducing base flipping and pseudobase-pair formation at the strand-exchanging points. Structures of MOC1 bound to HJ and different metal ions support a two-metal ion catalysis mechanism. Further molecular dynamics simulations and biochemical analyses reveal a communication between specific substrate recognition and metal ion-dependent catalysis. Our study thus provides a mechanism for how a resolvase turns substrate specificity into catalytic efficiency.

A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1.

GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.

GEN1 Endonuclease: Purification and Nuclease Assays.

Successful chromosome segregation depends on the timely removal of DNA recombination and replication intermediates that interlink sister chromatids. These intermediates are acted upon by structure-selective endonucleases that promote incisions close to the junction point. GEN1, a member of the Rad2/XPG endonuclease family, was identified on the basis of its ability to cleave Holliday junction recombination intermediates. Resolution occurs by a nick and counter-nick mechanism in strands that are symmetrically related across the junction point, leading to the formation of ligatable nicked duplex products. The actions of GEN1 are, however, not restricted to HJs, as 5'-flaps and replication fork structures also serve as excellent in vitro substrates for the nuclease. In the cellular context, GEN1 activity is observed late in the cell cycle, as most of the protein is excluded from the nucleus, such that it gains access to DNA intermediates after the breakdown of nuclear envelope. Nuclear exclusion ensures the protection of replication forks and other DNA secondary structures important for normal metabolic processes. In this chapter, we describe the purification of recombinant GEN1 and detail biochemical assays involving the use of synthetic DNA substrates and cruciform-containing plasmids.

Preparation and Resolution of Holliday Junction DNA Recombination Intermediates.

Holliday junctions provide a covalent link between recombining DNA molecules and need to be removed prior to chromosome segregation at mitosis. Defects in their resolution lead to mitotic catastrophe, characterized by the formation of DNA breaks and chromosome aberrations. Enzymes that resolve recombination intermediates have been identified in all forms of life, from bacteriophage, to bacteria, yeast, and humans. In higher eukaryotes, Holliday junctions are resolved by GEN1, a nuclease that is mechanistically similar to the prototypic resolvase Escherichia coli RuvC, and by the SMX trinuclease complex. Studies of these enzymes have been facilitated by the use of plasmid-sized DNA recombination intermediates made by RecA-mediated strand exchange. Here, we detail the preparation of these recombination intermediates, which resemble α-structures, and their resolution by RuvC and GEN1.

Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes.

Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.

Resolution of single and double Holliday junction recombination intermediates by GEN1.

Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5'-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5'-A/TTTG/C-3'.

Holliday junction-resolving enzymes-structures and mechanisms.

Holliday junction-resolving enzymes are nucleases that are highly specific for the structure of the junction, to which they bind in dimeric form. Two symmetrically disposed cleavages are made. These are not simultaneous, but the second cleavage is accelerated relative to the first, so ensuring that bilateral cleavage occurs during the lifetime of the DNA-protein complex. In eukaryotic cells there are two known junction-resolving activities. GEN1 is similar to enzymes from lower organisms. A crystallographic structure of a fungal GEN1 bound to the product of resolution has been determined. These complexes are dimerized within the crystal lattice such that the strands of the products may be simply reconnected to form a junction. These structures suggest a trajectory for the resolution process.

Structural insights into dynamics of RecU-HJ complex formation elucidates key role of NTR and stalk region toward formation of reactive state.

Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability. Further, we performed molecular dynamics (MD) simulations of wild type and an NTR deletion variant of RecU, with and without HJ. The NTR is observed to be highly flexible in simulations of the unbound RecU, in agreement with SAXS observations. These simulations revealed domain dynamics of RecU and their role in the formation of complex with HJ. The MD simulations also elucidate key roles of the NTR, stalk region, and breathing motion of RecU in the formation of the reactive state.

Structure and Metal Binding Properties of a Poxvirus Resolvase.

Poxviruses replicate their linear genomes by forming concatemers that must be resolved into monomeric units to produce new virions. A viral resolvase cleaves DNA four-way junctions extruded at the concatemer junctions to produce monomeric genomes. This cleavage reaction is required for viral replication, so the resolvase is an attractive target for small molecule inhibitors. To provide a platform for understanding resolvase mechanism and designing inhibitors, we have determined the crystal structure of the canarypox virus (CPV) resolvase. CPV resolvase is dimer of RNase H superfamily domains related to Escherichia coli RuvC, with an active site lined by highly conserved acidic residues that bind metal ions. There are several intriguing structural differences between resolvase and RuvC, and a model of the CPV resolvase·Holliday junction complex provides insights into the consequences of these differences, including a plausible explanation for the weak sequence specificity exhibited by the poxvirus enzymes. The model also explains why the poxvirus resolvases are more promiscuous than RuvC, cleaving a variety of branched, bulged, and flap-containing substrates. Based on the unique active site structure observed for CPV resolvase, we have carried out a series of experiments to test divalent ion usage and preferences. We find that the two resolvase metal binding sites have different preferences for Mg(2+) versus Mn(2+) Optimal resolvase activity is maintained with 5 µm Mn(2+) and 100 µm Mg(2+), concentrations that are well below those required for either metal alone. Together, our findings provide biochemical insights and structural models that will facilitate studying poxvirus replication and the search for efficient poxvirus inhibitors.

Crystal Structure of a Eukaryotic GEN1 Resolving Enzyme Bound to DNA.

We present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction.

Holliday junction resolvases.

Four-way DNA intermediates, called Holliday junctions (HJs), can form during meiotic and mitotic recombination, and their removal is crucial for chromosome segregation. A group of ubiquitous and highly specialized structure-selective endonucleases catalyze the cleavage of HJs into two disconnected DNA duplexes in a reaction called HJ resolution. These enzymes, called HJ resolvases, have been identified in bacteria and their bacteriophages, archaea, and eukaryotes. In this review, we discuss fundamental aspects of the HJ structure and their interaction with junction-resolving enzymes. This is followed by a brief discussion of the eubacterial RuvABC enzymes, which provide the paradigm for HJ resolvases in other organisms. Finally, we review the biochemical and structural properties of some well-characterized resolvases from archaea, bacteriophage, and eukaryotes.

FAN1 activity on asymmetric repair intermediates is mediated by an atypical monomeric virus-type replication-repair nuclease domain.

FAN1 is a structure-selective DNA repair nuclease with 5' flap endonuclease activity, involved in the repair of interstrand DNA crosslinks. It is the only eukaryotic protein with a virus-type replication-repair nuclease ("VRR-Nuc") "module" that commonly occurs as a standalone domain in many bacteria and viruses. Crystal structures of three representatives show that they structurally resemble Holliday junction resolvases (HJRs), are dimeric in solution, and are able to cleave symmetric four-way junctions. In contrast, FAN1 orthologs are monomeric and cleave 5' flap structures in vitro, but not Holliday junctions. Modeling of the VRR-Nuc domain of FAN1 reveals that it has an insertion, which packs against the dimerization interface observed in the structures of the viral/bacterial VRR-Nuc proteins. We propose that these additional structural elements in FAN1 prevent dimerization and bias specificity toward flap structures.

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 RuvC resolvase in complex with Holliday junction substrate.

The key intermediate in genetic recombination is the Holliday junction (HJ), a four-way DNA structure. At the end of recombination, HJs are cleaved by specific nucleases called resolvases. In Gram-negative bacteria, this cleavage is performed by RuvC, a dimeric endonuclease that belongs to the retroviral integrase superfamily. Here, we report the first crystal structure of RuvC in complex with a synthetic HJ solved at 3.75 Å resolution. The junction in the complex is in an unfolded 2-fold symmetrical conformation, in which the four arms point toward the vertices of a tetrahedron. The two scissile phosphates are located one nucleotide from the strand exchange point, and RuvC approaches them from the minor groove side. The key protein-DNA contacts observed in the structure were verified using a thiol-based site-specific cross-linking approach. Compared with known complex structures of the phage resolvases endonuclease I and endonuclease VII, the RuvC structure exhibits striking differences in the mode of substrate binding and location of the cleavage site.