Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family

The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5′-CCGC/GG-3′ (‘/’ indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺, Cu²⁺ and Ca²⁺. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.     

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.The UL24 protein is conserved throughout the Herpesviridae family, and to the best of our knowledge, a UL24 homolog has been identified in all Herpesvirales genomes sequenced to date with the exception of the channel catfish virus (9, 10, 19). UL24 of herpes simplex virus 1 (HSV-1) is required for efficient virus replication both in vitro and in vivo and for reactivation from latency in a mouse model of ocular infection (18). UL24 is one of the few HSV-1 genes, along with gB, gK, and UL20, in which mutations have been identified that cause the formation of syncytial plaques (2, 7, 34, 36, 39). The UL24-associated syncytial phenotype is only partially penetrant at 37°C but is fully penetrant at 39°C. Indications are that gK and UL20 have an inhibitory effect on the formation of syncytia (1), while certain mutations in gB entrain an uncontrolled fusogenic activity (11, 13, 15).UL24 is a highly basic protein of 269 amino acids that is expressed with leaky-late kinetics (31). Five homology domains (HDs), which consist of stretches of amino acids with a high percentage of identity between homologs, are present in the UL24 open reading frame (ORF) (19). In addition, a PD-(D/E)XK endonuclease motif has been identified that falls within the HDs (20); however, a role for this motif has yet to be demonstrated. In infected cells, UL24 is detected in the nucleus and the cytoplasm and transiently localizes to nucleoli (23). In the absence of other viral proteins, UL24 accumulates in the Golgi apparatus and in the nucleus, where it usually exhibits a diffuse staining pattern, but in a minority of cells it is detected in nucleoli (3).During infection, the formation of the viral replication compartments in the nucleus and the action of several viral proteins result in a remodeling of the nucleus. Chromatin is marginalized (29, 40), promyelocytic leukemia bodies are dispersed (26, 27), and the nuclear lamina is disrupted (33, 37). HSV-1 infection also affects the nucleolus, a prominent nuclear substructure implicated in the synthesis of rRNA, cell cycle regulation, and nucleocytoplasmic shuttling (5). Nucleoli become elongated following infection, and the synthesis of mature rRNA is reduced (4, 38, 42). Several HSV-1 proteins have been shown to localize to, or associate with, the nucleolus (12). The viral protein VP22 associates with the nucleolus and with dispersed nucleolin in HSV-1-infected cells (22), and RL1, US11, and ICP0 have also been shown to localize to nucleoli (24, 30, 35). Previously we showed that nucleolin is dispersed throughout the nucleus upon HSV-1 infection and that UL24 is involved in this nuclear modification (23). We further found that the N-terminal portion of UL24 is sufficient to induce the redistribution of nucleolin in the absence of other viral proteins (3).In this study, we sought to test the hypothesis that the endonuclease motif, which is made up of some of the most highly conserved residues in UL24, is important for the dispersal of nucleolin. A panel of substitution mutations in UL24 was generated, and the impact on the function of UL24 was assessed.     

Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches

Background  

PD-(D/E)XK nucleases constitute a large and highly diverse superfamily of enzymes that display little sequence similarity despite retaining a common core fold and a few critical active site residues. This makes identification of new PD-(D/E)XK nuclease families a challenging task as they usually escape detection with standard sequence-based methods. We developed a modified transitive meta profile search approach and to consider the structural diversity of PD-(D/E)XK nuclease fold more thoroughly we analyzed also lower than threshold Meta-BASIC hits to select potentially correct predictions placed among unreliable or incorrect ones.     

Activity and Specificity of the Bacterial PD-(D/E)XK Homing Endonuclease I-Ssp6803I

The restriction endonuclease fold [a three-layer α-β sandwich containing variations of the PD-(D/E)XK nuclease motif] has been greatly diversified during evolution, facilitating its use for many biological functions. Here we characterize DNA binding and cleavage by the PD-(D/E)XK homing endonuclease I-Ssp6803I. Unlike most restriction endonucleases harboring the same core fold, the specificity profile of this enzyme extends over a long (17 bp) target site. The DNA binding and cleavage specificity profiles of this enzyme were independently determined and found to be highly correlated. However, the DNA target sequence contains several positions where binding and cleavage activities are not tightly coupled: individual DNA base-pair substitutions at those positions that significantly decrease cleavage activity have minor effects on binding affinity. These changes in the DNA target sequence appear to correspond to substitutions that uniquely increase the free energy change between the ground state and the transition state, rather than simply decreasing the overall DNA binding affinity. The specificity of the enzyme reflects constraints on its host gene and limitations imposed by the enzyme's quaternary structure and illustrate the highly diverse repertoire of DNA recognition specificities that can be adopted by the related folds surrounding the PD-(D/E)XK nuclease motif.     

I-Ssp6803I: the first homing endonuclease from the PD-(D/E)XK superfamily exhibits an unusual mode of DNA recognition

MOTIVATION: Restriction endonucleases (REases) and homing endonucleases (HEases) are biotechnologically important enzymes. Nearly all structurally characterized REases belong to the PD-(D/E)XK superfamily of nucleases, while most HEases belong to an unrelated LAGLIDADG superfamily. These two protein folds are typically associated with very different modes of protein-DNA recognition, consistent with the different mechanisms of action required to achieve high specificity. REases recognize short DNA sequences using multiple contacts per base pair, while HEases recognize very long sites using a few contacts per base pair, thereby allowing for partial degeneracy of the target sequence. Thus far, neither REases with the LAGLIDADG fold, nor HEases with the PD-(D/E)XK fold, have been found. RESULTS: Using protein fold recognition, we have identified the first member of the PD-(D/E)XK superfamily among homing endonucleases, a cyanobacterial enzyme I-Ssp6803I. We present a model of the I-Ssp6803I-DNA complex based on the structure of Type II restriction endonuclease R.BglI and predict the active site and residues involved in specific DNA sequence recognition by I-Ssp6803I. Our finding reveals a new unexpected evolutionary link between HEases and REases and suggests how PD-(D/E)XK nucleases may develop a 'HEase-like' way of interacting with the extended DNA sequence. This in turn may be exploited to study the evolution of DNA sequence specificity and to engineer nucleases with new substrate specificities.     

Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily

Proteins belonging to PD-(D/E)XK phosphodiesterases constitute a functionally diverse superfamily with representatives involved in replication, restriction, DNA repair and tRNA–intron splicing. Their malfunction in humans triggers severe diseases, such as Fanconi anemia and Xeroderma pigmentosum. To date there have been several attempts to identify and classify new PD-(D/E)KK phosphodiesterases using remote homology detection methods. Such efforts are complicated, because the superfamily exhibits extreme sequence and structural divergence. Using advanced homology detection methods supported with superfamily-wide domain architecture and horizontal gene transfer analyses, we provide a comprehensive reclassification of proteins containing a PD-(D/E)XK domain. The PD-(D/E)XK phosphodiesterases span over 21 900 proteins, which can be classified into 121 groups of various families. Eleven of them, including DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII and Replic_Relax, are newly assigned to the PD-(D/E)XK superfamily. Some groups of PD-(D/E)XK proteins are present in all domains of life, whereas others occur within small numbers of organisms. We observed multiple horizontal gene transfers even between human pathogenic bacteria or from Prokaryota to Eukaryota. Uncommon domain arrangements greatly elaborate the PD-(D/E)XK world. These include domain architectures suggesting regulatory roles in Eukaryotes, like stress sensing and cell-cycle regulation. Our results may inspire further experimental studies aimed at identification of exact biological functions, specific substrates and molecular mechanisms of reactions performed by these highly diverse proteins.     

Identification of new homologs of PD-(D/E)XK nucleases by support vector machines trained on data derived from profile-profile alignments

PD-(D/E)XK nucleases, initially represented by only Type II restriction enzymes, now comprise a large and extremely diverse superfamily of proteins. They participate in many different nucleic acids transactions including DNA degradation, recombination, repair and RNA processing. Different PD-(D/E)XK families, although sharing a structurally conserved core, typically display little or no detectable sequence similarity except for the active site motifs. This makes the identification of new superfamily members using standard homology search techniques challenging. To tackle this problem, we developed a method for the detection of PD-(D/E)XK families based on the binary classification of profile-profile alignments using support vector machines (SVMs). Using a number of both superfamily-specific and general features, SVMs were trained to identify true positive alignments of PD-(D/E)XK representatives. With this method we identified several PFAM families of uncharacterized proteins as putative new members of the PD-(D/E)XK superfamily. In addition, we assigned several unclassified restriction enzymes to the PD-(D/E)XK type. Results show that the new method is able to make confident assignments even for alignments that have statistically insignificant scores. We also implemented the method as a freely accessible web server at http://www.ibt.lt/bioinformatics/software/pdexk/.     

An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI

Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction–modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.     

Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases

The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.     

The PD-(D/E)XK superfamily revisited: identification of new members among proteins involved in DNA metabolism and functional predictions for domains of (hitherto) unknown function

Background  

The PD-(D/E)XK nuclease superfamily, initially identified in type II restriction endonucleases and later in many enzymes involved in DNA recombination and repair, is one of the most challenging targets for protein sequence analysis and structure prediction. Typically, the sequence similarity between these proteins is so low, that most of the relationships between known members of the PD-(D/E)XK superfamily were identified only after the corresponding structures were determined experimentally. Thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional PSI-BLAST searches.     

Grouping together highly diverged PD-(D/E)XK nucleases and identification of novel superfamily members using structure-guided alignment of sequence profiles

The PD-(D/E)XK nuclease domains, initially identified in type II restriction enzymes, serve as models for studying aspects of protein-DNA interactions, mechanisms of phosphodiester hydrolysis, and provide indispensable tools for techniques in genetic engineering and molecular medicine. However, the low degree of amino acid conservation hampers the possibility of identification of PD-(D/E)XK superfamily members based solely on sequence comparisons. In several proteins implicated in DNA recombination and repair the restriction enzyme-like nuclease domain has been found only after the corresponding structures were determined experimentally. Here, we identified highly diverged variants of the PD-(D/E)XK domain in many proteins and open reading frames using iterative database searches and progressive, structure-guided alignment of sequence profiles. We predicted the possible cellular function for many hypothetical proteins based on their relative similarity to characterized nucleases or observed presence of additional domains. We also identified the nuclease domain in genuine recombinases and restriction enzymes, whose homology to other PD-(D/E)XK enzymes has not been demonstrated previously. The first superfamily-wide comparative analysis, not limited to nucleases of known structure, will guide cloning and characterization of novel enzymes and planning new experiments to better understand those already studied.     

Structure of Xanthomonas axonopodis pv. citri YaeQ reveals a new compact protein fold built around a variation of the PD-(D/E)XK nuclease motif

The YaeQ family of proteins are found in many Gram-negative and a few Gram-positive bacteria. We have determined the first structure of a member of the YaeQ family by X-ray crystallography. Comparisons with other structures indicate that YaeQ represents a new compact protein fold built around a variation of the PD-(D/E)XK nuclease motif found in type II endonucleases and enzymes involved in DNA replication, repair, and recombination. We show that catalytically important residues in the PD-(D/E)XK nuclease superfamily are spatially conserved in YaeQ and other highly conserved YaeQ residues may be poised to interact with nucleic acid structures.     

BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.     

DNA intercalation without flipping in the specific ThaI–DNA complex

The PD-(D/E)XK type II restriction endonuclease ThaI cuts the target sequence CG/CG with blunt ends. Here, we report the 1.3 Å resolution structure of the enzyme in complex with substrate DNA and a sodium or calcium ion taking the place of a catalytic magnesium ion. The structure identifies Glu54, Asp82 and Lys93 as the active site residues. This agrees with earlier bioinformatic predictions and implies that the PD and (D/E)XK motifs in the sequence are incidental. DNA recognition is very unusual: the two Met47 residues of the ThaI dimer intercalate symmetrically into the CG steps of the target sequence. They approach the DNA from the minor groove side and penetrate the base stack entirely. The DNA accommodates the intercalating residues without nucleotide flipping by a doubling of the CG step rise to twice its usual value, which is accompanied by drastic unwinding. Displacement of the Met47 side chains from the base pair midlines toward the downstream CG steps leads to large and compensating tilts of the first and second CG steps. DNA intercalation by ThaI is unlike intercalation by HincII, HinP1I or proteins that bend or repair DNA.     

Identification of a PD-(D/E)XK-like domain with a novel configuration of the endonuclease active site in the methyl-directed restriction enzyme Mrr and its homologs

The Escherichia coli K-12 restriction enzyme Mrr recognizes and cleaves N6-methyladenine- and 5-methylcytosine-containing DNA. Its amino acid sequence has been subjected to structure prediction and comparison with other sequences from publicly available sources. The results obtained suggest that Mrr and related putative endonucleases possess a cleavage domain typical for all the so far structurally characterized type II restriction enzymes, however with an unusual glutamine residue at the central position of the (D/E)-(D/E)XK hallmark of the active site. The "missing" acidic side chain was instead found anchored in a different, unusual position, suggesting that Mrr represents a third topological variant of the endonuclease active site in addition to the two alternatives determined previously (Skirgaila et al., 1998. J. Mol. Biol. 279, 473-481). One of the newly identified putative endonucleases from the Mrr family is composed of two diverged cleavage domains, which possess both the "typical" D-EXK and the "Mrr-like" D-QXK variants of the active site. Among the Mrr homologs there are also proteins from yeast, in which restriction phenotype has not been observed, suggesting that the free-standing Eukaryotic PD-(D/E)XK superfamily members might be implicated in other cellular processes involving enzymatic DNA cleavage.     

Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit

The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.     

The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.     

Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima

Xylulokinase (XK, E.C. 2.7.1.17) is one of the key enzymes in xylose metabolism and it is essential for the activation of pentoses for the sustainable production of biocommodities from biomass sugars. The open reading frame (TM0116) from the hyperthermophilic bacterium Thermotoga maritima MSB8 encoding a putative xylulokinase were cloned and expressed in Escherichia coli BL21 Star (DE3) in the Luria–Bertani and auto-inducing high-cell-density media. The basic biochemical properties of this thermophilic XK were characterized. This XK has the optimal temperature of 85 °C. Under a suboptimal condition of 60 °C, the k _cat was 83 s^?1, and the K _m values for xylulose and ATP were 1.24 and 0.71 mM, respectively. We hypothesized that this XK could work on polyphosphate possibly because this ancestral thermophilic microorganism utilizes polyphosphate to regulate the Embden–Meyerhof pathway and its substrate-binding residues are somewhat similar to those of other ATP/polyphosphate-dependent kinases. This XK was found to work on low-cost polyphosphate, exhibiting 41 % of its specific activity on ATP. This first ATP/polyphosphate XK could have a great potential for xylose utilization in thermophilic ethanol-producing microorganisms and cell-free biosystems for low-cost biomanufacturing without the use of ATP.