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.     

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.     

Protein stability indicates divergent evolution of PD-(D/E)XK type II restriction endonucleases

Type II restriction endonucleases recognize 4-8 base-pair-long DNA sequences and catalyze their cleavage with remarkable specificity. Crystal structures of the PD-(DE)XK superfamily revealed a common alpha/beta core motif and similar active site. In contrast, these enzymes show little sequence similarity and use different strategies to interact with their substrate DNA. The intriguing question is whether this enzyme family could have evolved from a common origin. In our present work, protein structure stability elements were analyzed and compared in three parts of PD-(DE)XK type II restriction endonucleases: (1) core motif, (2) active-site residues, and (3) residues playing role in DNA recognition. High correlation was found between the active-site residues and those stabilization factors that contribute to preventing structural decay. DNA recognition sites were also observed to participate in stabilization centers. It indicates that recognition motifs and active sites in PD-(DE)XK type II restriction endonucleases should have been evolutionary more conserved than other parts of the structure. Based on this observation it is proposed that PD-(DE)XK type II restriction endonucleases have developed from a common ancestor with divergent evolution.     

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.     

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.     

Human herpesvirus 8 gene encodes a functional thymidylate synthase

We demonstrate that human herpesvirus 8, obtained from the lymphoma cell line BC-3 as well as from Kaposi's sarcoma lesions, carries a gene that encodes a functional thymidylate synthase (TS). The particular characteristics of this enzyme are studied and compared to the characteristics of TSs encoded by other organisms.     

Human xeroderma pigmentosum group G gene encodes a DNA endonuclease.

Because of defective nucleotide excision repair of ultraviolet damaged DNA, xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers. Cell fusion studies have identified seven XP complementation groups, A to G. Previous studies have implicated the products of these seven XP genes in the recognition of ultraviolet-induced DNA damage and in incision of the damage-containing DNA strand. Here, we express the XPG-encoded protein in Sf9 insect cells and purify it to homogeneity. We demonstrate that XPG is a single-strand specific DNA endonuclease, thus identifying the catalytic role of the protein in nucleotide excision repair. We suggest that XPG nuclease acts on the single-stranded region created as a result of the combined action of the XPB helicase and XPD helicase at the DNA damage site.     

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.     

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.     

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.     

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/.     

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.     

Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein.

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.     

The equine herpesvirus 2 E1 open reading frame encodes a functional chemokine receptor

Several herpesviruses contain open reading frames (ORFs) that encode potential homologs of eucaryotic genes. Equine herpesvirus 2 (EHV-2) is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. The E1 ORF of EHV-2, a G protein-coupled receptor homolog, shows 31 to 47% amino acid identity with known CC chemokine receptors. To investigate whether E1 may encode a functional receptor, we cloned the E1 ORF and expressed it in stably transfected cell lines. We report here the identification of the CC chemokine eotaxin as a functional ligand for the EHV-2 E1 receptor. Chemokines are likely to play a role in the regulation of immune functions in equine hosts during EHV-2 infection and, via interaction with E1, may affect viral replication and/or escape from immune responses.     

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.     

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.     

The human cytomegalovirus-specific UL1 gene encodes a late-phase glycoprotein incorporated in the virion envelope

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.     

The Caenorhabditis elegans gene CeAPN1 encodes a homolog of Escherichia coli and yeast apurinic/apyrimidinic endonuclease

The Saccharomyces cerevisiae APNI gene, encoding the bifunctional DNA repair enzyme apurinic/apyrimidinic (AP) endonuclease/3′-repair diesterase, was used as a probe to isolate a gene homolog, CeAPN1, from a Caenorhabditis elegans cDNA library. The CeAPN1 gene is predicted to encode a protein 30 kDa in size, which shares 40.4% and 44.9% identity at the amino acid level with, respectively, S. cerevisiae Apnl and Escherichia coli endonuclease IV. We suggest that CeApn1 protein is a member of the endonuclease IV family of DNA repair enzymes.