Holliday Junction Resolvase Research Articles

Holliday Junction Resolution.

Holliday junctions are four-way DNA structures that may arise during meiotic recombination, double-strand break repair, or postreplicative repair by the reciprocal exchange of single strands between two DNA molecules. Given their ability to effectively bridge two sister chromatids or homologous chromosomes, cells have implemented various pathways to ensure their timely removal. One of them is the nucleolytic processing of the Holliday junctions by specialized structure-selective endonucleases termed resolvases, which sever the connection between the linked molecules. These Holliday junction resolvases are essential tools of the DNA damage repair machinery to ensure accurate chromosomal segregation, whose activities can be modulated by posttranslational modifications like phosphorylation. Here, we describe a protocol to purify S. cerevisiae Yen1 resolvase in two different phosphorylation states (high and low) and to set up a biochemical assay to compare their ability to process a synthetic, oligonucleotide-based Holliday junction structures.

Structural insights into sequence-dependent Holliday junction resolution by the chloroplast resolvase MOC1

Holliday junctions (HJs) are key DNA intermediates in genetic recombination and are eliminated by nuclease, termed resolvase, to ensure genome stability. HJ resolvases have been identified across all kingdoms of life, members of which exhibit sequence-dependent HJ resolution. However, the molecular basis of sequence selectivity remains largely unknown. Here, we present the chloroplast resolvase MOC1, which cleaves HJ in a cytosine-dependent manner. We determine the crystal structure of MOC1 with and without HJs. MOC1 exhibits an RNase H fold, belonging to the retroviral integrase family. MOC1 functions as a dimer, and the HJ is embedded into the basic cleft of the dimeric enzyme. We characterize a base recognition loop (BR loop) that protrudes into and opens the junction. Residues from the BR loop intercalate into the bases, disrupt the C-G base pairing at the crossover and recognize the cytosine, providing the molecular basis for sequence-dependent HJ resolution by a resolvase.

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.

Gen1 mutation caused kidney hypoplasia and defective ureter-bladder connections in mice.

Limited genetic factors were uncovered for the development of congenital anomalies of the kidney and urinary tract (CAKUT). We previously reported that a Holliday junction resolvase Gen1 was essential for early metanephric development in mice. This comprehensive follow-up study focused on the roles of Gen1 in late metanephric development. We found that Gen1 mutation impaired the late development of both kidney and urinary tract. In vivo and ex-vivo kidney primordia culture confirmed decreased ureteric bud branching in Gen1 mutants, which consequently caused hypoplasia. We also observed abnormal urinary tract development. Programmed apoptosis at the end of nephric duct disappeared in Gen1 mutants, which caused abnormal ureter-bladder connections, leading to vesicoureteral reflux (VUR) or ureterovesical junction obstruction (UVJO). Mechanistically, RNA-seq analysis proved that Gen1 mutation impaired the expression of multiple regulatory genes for the metanephric development, including Six2. Taken together, our study provides more insight into the roles of Gen1 in the development of the kidney and urinary tract, which may have potential clinical significance in the treatment and/or prevention of CAKUT.

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.

Exploring alternative catalytic mechanisms of the Cas9 HNH domain.

Understanding the reaction mechanism of CRISPR-associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo- and substrate-bound forms, the catalytically active structure is still unclear. Our first attempt to explore the catalytic mechanism of Cas9 HNH domain has been based on the reasonable assumption that we are dealing with the same mechanism as endonuclease VII, including the assumption that the catalytic water is in the first shell of the Mg2+ . Trying this mechanism with the cryo-EM structure forced us to induce significant structural change driven by the movement of K848 (or other positively charged residue) close to the active site to facilitate the proton transfer step. In the present study, we explore a second reaction mechanism where the catalytic water is in the second shell of the Mg2+ and assume that the cryo-EM structure by itself is a suitable representation of a catalytic-ready structure. The alternative mechanism indicates that if the active water is from the second shell, then the calculated reaction barrier is lower compared with the corresponding barrier when the water comes from the first shell.

Nuclear and cytoplasmic WDR-23 isoforms mediate differential effects on GEN-1 and SKN-1 substrates

Maintaining a healthy cellular environment requires the constant control of proteostasis. E3 ubiquitin ligase complexes facilitate the post-translational addition of ubiquitin, which based on the quantity and specific lysine linkages, results in different outcomes. Our studies reveal the CUL4-DDB1 substrate receptor, WDR23, as both a positive and a negative regulator in cellular stress responses. These opposing roles are mediated by two distinct isoforms: WDR-23A in the cytoplasm and WDR-23B in the nucleus. C. elegans expressing only WDR-23A display activation of SKN-1 and enhanced survival to oxidative stress, whereas animals with restricted WDR-23B expression do not. Additionally, we identify GEN-1, a Holliday junction resolvase, as an evolutionarily conserved WDR-23 substrate and find that the nuclear and cytoplasmic isoforms of WDR-23 differentially affect double-strand break repair. Our results suggest that through differential ubiquitination, nuclear WDR-23B inhibits the activity of substrates, most likely by promoting protein turnover, while cytoplasmic WDR-23A performs a proteasome-independent role. Together, our results establish a cooperative role between two spatially distinct isoforms of WDR-23 in ensuring proper regulation of WDR-23 substrates.

Phosphorylation of the Archaeal Holliday Junction Resolvase Hjc Inhibits Its Catalytic Activity and Facilitates DNA Repair in Sulfolobus islandicus REY15A.

Protein phosphorylation is one of the main protein post-translational modifications and regulates DNA repair in eukaryotes. Archaeal genomes encode eukaryotic-like DNA repair proteins and protein kinases (ePKs), and several proteins involved in homologous recombination repair (HRR) including Hjc, a conserved Holliday junction (HJ) resolvase in Archaea, undergo phosphorylation, indicating that phosphorylation plays important roles in HRR. Herein, we performed phosphorylation analysis of Hjc by various ePKs from Sulfolobus islandicus. It was shown that SiRe_0171, SiRe_2030, and SiRe_2056, were able to phosphorylate Hjc in vitro. These ePKs phosphorylated Hjc at different Ser/Thr residues: SiRe_0171 on S34, SiRe_2030 on both S9 and T138, and SiRe_2056 on T138. The HJ cleavage activity of the phosphorylation-mimic mutants was analyzed and the results showed that the cleavage activity of S34E was completely lost and that of S9E had greatly reduced. S. islandicus strain expressing S34E in replacement of the wild type Hjc was resistant to higher doses of DNA damaging agents. Furthermore, SiRe_0171 deletion mutant exhibited higher sensitivity to DNA damaging agents, suggesting that Hjc phosphorylation by SiRe_0171 enhanced the DNA repair capability. Our results revealed that HJ resolvase is regulated by protein phosphorylation, reminiscent of the regulation of eukaryotic HJ resolvases GEN1 and Yen1.

Helicase/SUMO-targeted ubiquitin ligase Uls1 interacts with the Holliday junction resolvase Yen1.

Resolution of branched DNA structures is pivotal for repair of stalled replication forks and meiotic recombination intermediates. The Yen1 nuclease cleaves both Holliday junctions and replication forks. We show that Yen1 interacts physically with Uls1, a suggested SUMO-targeted ubiquitin ligase that also contains a SWI/SNF-family ATPase-domain. Yen1 is SUMO-modified in its noncatalytic carboxyl terminus and DNA damage induces SUMOylation. SUMO-modification of Yen1 strengthens the interaction to Uls1, and mutations in SUMO interaction motifs in Uls1 weakens the interaction. However, Uls1 does not regulate the steady-state level of SUMO-modified Yen1 or chromatin-associated Yen1. In addition, SUMO-modification of Yen1 does not affect the catalytic activity in vitro. Consistent with a shared function for Uls1 and Yen1, mutations in both genes display similar phenotypes. Both uls1 and yen1 display negative genetic interactions with the alternative HJ-cleaving nuclease Mus81, manifested both in hypersensitivity to DNA damaging agents and in meiotic defects. Point mutations in ULS1 (uls1K975R and uls1C1330S, C1333S) predicted to inactivate the ATPase and ubiquitin ligase activities, respectively, are as defective as the null allele, indicating that both functions of Uls1 are essential. A micrococcal nuclease sequencing experiment showed that Uls1 had minimal effects on global nucleosome positioning/occupancy. Moreover, increased gene dosage of YEN1 partially alleviates the mus81 uls1 sensitivity to DNA damage. We suggest a preliminary model in which Uls1 acts in the same pathway as Yen1 to resolve branched DNA structures.

Structural and Functional Insights into CRISPR/Cas9 Catalytic Activation and Specificity Enhancement

CRISPR/Cas9 has been repurposed as a powerful genome editing tool, with immense potential toward therapeutic applications. Despite recent advances in understanding DNA recognition and cleavage by Cas9, consistent structural and functional information about the catalytic state of the HNH nuclease domain remains to be elucidated. On the basis of our recent work (Nature Scientific Reports, 7:17271), we here report a new Cas9 active state complex structure discovered by molecular dynamics simulations and validated by site-directed mutagenesis experiments. In this structure, the HNH domain is poised for cleaving the target DNA strand using a canonical catalytic triad as seen in phage T4 Endonuclease VII. Guided by the derived new structural information, we rationally designed and tested a library of new Cas9 variants to improve Cas9 specificities. Our ultimate goal is to offer several high-fidelity Cas9 enzymes that can be broadly used in basic research and medical therapy.

Freedom of movement.

Holliday junction resolvases lock dynamic DNA four-way junctions into specific structural conformations for symmetric DNA cleavage. Single-molecule studies now reveal that resolvases can relax their grip, enabling Holliday junction conformer transitions and branch migration in the enzyme-bound form.

Gen1 Modulates Metanephric Morphology Through Retinoic Acid Signaling.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of end-stage renal disease in children. Our group has discovered that Holliday Junction resolvase gene Gen1 is a potential candidate gene for CAKUT. Gen1 mutant mice showed CAKUT phenotypes similar to those observed in retinoic acid (RA)-deficient models. The expression of Raldh2, which encodes the key enzyme in RA synthesis, was reduced in Gen1 mutant metanephros through RNA sequencing. By real-time reverse transcription-PCR, the expression of both Raldh2 and downstream Ret was reduced in embryonic day (E) 11.5 Gen1 mutant ureters and E13.5 kidneys, and expression of RA receptor alpha was decreased in E13.5 Gen1 mutant ureters and kidneys. Further studies showed that all-trans retinoic acid (ATRA) rescued solitary kidney phenotype and improved ureteric branching; ATRA should be administered after ureteric budding to avoid increasing the incidence of ectopic budding in Gen1 mutants. Luciferase intensity of RA response element was lower in CHO-K1 cells transfected with Gen1 siRNA than in those transfected with scrambled RNA, and this inhibitory effect could be reversed by ATRA. These findings indicate that Gen1 mutation can result in renal malformation through RA signaling and Gen1-loss-induced CAKUT can be partly rescued by ATRA.

Exploring the Catalytic Mechanism of Cas9 Using Information Inferred from Endonuclease VII.

Elucidating the nature of the gene editing mechanism of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an important task in view of the role of this breakthrough to the advancement of human medicine. In particular, it is crucial to understand the catalytic mechanism of Cas9 (one of the CRISPR associated proteins) and its role in confirming accurate editing. Thus, we focus in this work on an attempt to analyze the catalytic mechanism of Cas9. Considering the absence of detailed structural information on the active form of Cas9, we use an empirical valence bond (EVB) which is calibrated on the closely related mechanism of T4 endonuclease VII. The calibrated EVB is then used in studying the reaction of Cas9, while trying several structural models. It is found that the catalytic activation requires a large conformational change, where K848 or other positively charged group moves from a relatively large distance toward the scissile phosphate. This conformational change leads to the change in position of the Mg2+ ion and to a major reduction in the activation barrier for the catalytic reaction. Our finding provides an important clue on the nature of the catalytic activation of CAS9 and thus should help in elucidating a key aspect of the gene editing process. For example, the approach used here should be effective in exploring the nature of off target activation and its relationship to the energetics of the unwinding process. This strategy may offer ways to improve the selectivity of Cas9.

Regulation of Structure-Specific Endonucleases in Replication Stress.

Replication stress results in various forms of aberrant replication intermediates that need to be resolved for faithful chromosome segregation. Structure-specific endonucleases (SSEs) recognize DNA secondary structures rather than primary sequences and play key roles during DNA repair and replication stress. Holliday junction resolvase MUS81 (methyl methane sulfonate (MMS), and UV-sensitive protein 81) and XPF (xeroderma pigmentosum group F-complementing protein) are a subset of SSEs that resolve aberrant replication structures. To ensure genome stability and prevent unnecessary DNA breakage, these SSEs are tightly regulated by the cell cycle and replication checkpoints. We discuss the regulatory network that control activities of MUS81 and XPF and briefly mention other SSEs involved in the resolution of replication intermediates.

The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection.

Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and, more recently, a second bacteriophage resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation detection.

F-CphI represents a new homing endonuclease family using the Endo VII catalytic motif

BackgroundThere are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Previously, we discovered a novel homing endonuclease F-CphI encoded by ORF177 of cyanophage S-PM2. F-CphI does not resemble any characterized homing endonucleases. Instead, the C-terminus of F-CphI aligns well with the N-terminal catalytic domain of a Holliday junction DNA resolvase, phage T4 endonuclease VII (Endo VII).ResultsA PSI-BLAST search resulted in a total of 313 Endo VII motif–containing sequences in sequenced genomes. Multiple sequence alignment showed that the catalytically important residues of T4 Endo VII were all well conserved in these proteins. Our site-directed mutagenesis studies further confirmed that the catalytically important residues of T4 Endo VII were also essential for F-CphI activity, and thus F-CphI might use a similar protein fold as Endo VII for DNA cleavage. A phylogenetic tree of the Endo VII motif–containing sequences showed that putative resolvases grouped into one clade while putative homing endonucleases and restriction endonucleases grouped into another clade.ConclusionsBased on the unique conserved residues, we proposed that F-CphI represents a new homing endonuclease family, which was named the DHHRN family. Our phylogenetic analysis could be used to predict the functions of many previously unknown proteins.

The protein-protein interactions between Amsacta moorei entomopoxvirus (AMEV) protein kinases (PKs) and all viral proteins

Entomopoxviruses are an important group of viruses infecting only insects. They belong to Poxviridae which infect both invertebrates and vertebrates, including humans. Protein kinases are known to have roles at virus morphogenesis, host selectivity, the regulation of cell division and apoptosis in some vertebrate poxviruses. In this study, 2 protein kinases (PKs) (AMV153 and AMV197) of Amsacta moorei entomopoxvirus (AMEV) were investigated for the interactions among 230 viral proteins using yeast two-hybrid system (Y2H). For this purpose, two protein kinases and 230 viral genes were cloned into the bait and prey vectors, respectively. Bait vectors were introduced into Saccharomyces cerevisiae AH109. Expression of the bait genes were confirmed by western blot analysis. Both yeast strains of bait were transformed individually with each prey clone and grown on a selective medium (minimal synthetic defined) to determine the protein–protein interactions between bait and prey proteins. Transformations identified totally 16 interactions among AMEV protein kinases and all viral proteins of which 5 belong to AMV153 and 11 belong to AMV197. One of the five interactions detected for AMV153 protein kinase is self-association. Its other four interactions are with two virus entry complex proteins (AMV035 and AMV083), a membrane protein (AMV165) and a subunit of RNA polymerase (AMV230). The other protein kinase, AMV197, interacted with two virus entry complex proteins (AMV035 and AMV083) as AMV153, a caspase-2 enzyme (AMV063), a Holliday junction resolvase (AMV162), a membrane protein (AMV165), a subunit of RNA polymerase (AMV230) and five other hypothetical proteins (AMV026, AMV040, AMV062, AMV069, AMV120) encoded by AMEV genome. Glutathione S-transferase (GST) pull-down assay was used to confirm all interactions described by Y2H analysis. In addition, the theoretical structures of the two of 16 interactions were interpreted by docking analysis. Consistent with Y2H and pull down assays, docking analysis also showed the interactions of AMV063 with AMV153 and AMV197. Detected interactions of the AMEV viral proteins with viral protein kinases could lead to the understanding of the regulation of the viral activities of interacted viral proteins.

Disruption of Gen1 Causes Congenital Anomalies of the Kidney and Urinary Tract in Mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) are among the most common developmental defects in humans. Despite of several known CAKUT-related loci (HNF1B, PAX2, EYA1, etc.), the genetic etiology of CAKUT remains to be elucidated for most patients. In this study, we report that disruption of the Holliday Junction resolvase gene Gen1 leads to renal agenesis, duplex kidney, hydronephrosis, and vesicoureteral reflux (VUR) in mice. GEN1 interacts with SIX1 and enhances the transcriptional activity of SIX1/EYA1, a key regulatory complex of the GDNF morphogen. Gen1 mutation impairs Grem1 and Gdnf expression, resulting in excessive ureteric bud formation and defective ureteric bud branching during early kidney development. These results revealed an unidentified role of GEN1 in kidney development and suggested its contribution to CAKUT.

Identification of Holliday junction resolvases crucial for the chloroplast nucleoid morphology and segregation

Identification of Holliday junction resolvases crucial for the chloroplast nucleoid morphology and segregation

Genotypic variations between wild-type and small colony variant of Staphylococcus aureus in prosthetic valve infectious endocarditis: a comparative genomic and transcriptomic analysis

Staphylococcus aureus small colony variants (SCVs) can cause persistent infections. However, the genomes and transcriptomes of S. aureus SCVs remain poorly understood. A pair of isogenic wild-type and SCV methicillin-resistant S. aureus (MRSA) strains (IE1 and IE2, respectively) were isolated from a patient with prosthetic valve infectious endocarditis. The SCV strain IE2 grew more slowly than the wild-type strain, and serum killing and mouse lethality assays revealed that the virulence of SCV strain IE2 was decreased. Whole-genome sequencing of the SCV and wild-type strains revealed 15 mutations in nine genes associated with metabolism, virulence and DNA repair, including serine/threonine-protein kinase PrkC (prkC), glycerol-3-phosphate acyltransferase (plsY), 2-deoxyribose-5-phosphate aldolase (deoC), extracellular adherence protein (eap), iron compound ABC uptake transporter substrate-binding protein (sstD), RecU Holliday junction resolvase (recU), excinuclease ABC subunit B (uvrB), type I restriction–modification system, M subunit (hsdM) and smooth muscle caldesmon. Sequencing of RNA transcripts revealed that expression levels of 321 genes were upregulated and 582 genes were downregulated in SCV strain IE2 compared with IE1. Most of the differentially expressed genes were involved in metabolism. Expression levels of several genes involved in the pathways to which plsY, deoC, eap and sstD belonged were changed, associated with the metabolism and virulence of S. aureus. In conclusion, the reduced growth rate and decreased virulence of MRSA SCV strains may be related to mutations in and downregulation of genes associated with metabolism and virulence, especially plsY, deoC, eap and sstD.