Purification of the novel endonuclease, Hpy188I, and cloning of its restriction-modification genes reveal evidence of its horizontal transfer to the Helicobacter pylori genome

We have isolated a novel restriction endonuclease, Hpy188I, from Helicobacter pylori strain J188. Hpy188I recognizes the unique sequence, TCNGA, and cleaves the DNA between nucleotides N and G in its recognition sequence to generate a one-base 3' overhang. Cloning and sequence analysis of the Hpy188I modification gene in strain J188 reveal that hpy188IM has a 1299-base pair (bp) open reading frame (ORF) encoding a 432-amino acid product. The predicted protein sequence of M.Hpy188I contains conserved motifs typical of aminomethyltransferases, and Western blotting indicates that it is an N-6 adenine methyltransferase. Downstream of hpy188IM is a 513-bp ORF encoding a 170-amino acid product, that has a 41-bp overlap with hpy188IM. The predicted protein sequence from this ORF matches the amino acid sequence obtained from purified Hpy188I, indicating that it encodes the endonuclease. The Hpy188I R-M genes are not present in either strain of H. pylori that has been completely sequenced but are found in two of 11 H. pylori strains tested. The significantly lower G + C content of the Hpy188I R-M genes implies that they have been introduced relatively recently during the evolution of the H. pylori genome.     

Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI

Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.     

Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNA

The ββα-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5′-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β4α2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββα-Me region of the second β4α2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β4α2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββα-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).     

Experimental evidence for a beta beta alpha-Me-finger nuclease motif to represent the active site of the caspase-activated DNase

The caspase-activated DNase (CAD) is an important nuclease involved in apoptotic DNA degradation. Results of a sequence comparison of CAD proteins with beta beta alpha-Me-finger nucleases in conjunction with a mutational and chemical modification analysis suggest that CAD proteins constitute a new family of beta beta alpha-Me-finger nucleases. Nucleases of this family have widely different functions but are characterized by a common active-site fold and similar catalytic mechanisms. According to our results and comparisons with related nucleases, the active site of CAD displays features that partly resemble those of the colicin E9 and partly those of the T4 endonuclease VII active sites. We suggest that the catalytic mechanism of CAD involves a conserved histidine residue, acting as a general base, and another histidine as well as an aspartic acid residue required for cofactor binding. Our findings provide a first insight into the likely active-site structure and catalytic mechanism of a nuclease involved in the degradation of chromosomal DNA during programmed cell death.     

The active site of Serratia endonuclease contains a conserved magnesium-water cluster.

Serratia endonuclease is an important member of a class of magnesium dependent nucleases that are widely distributed in nature. Here, we describe the location and geometry of a magnesium-water cluster within the active site of this enzyme. The sole protein ligand of the magnesium atom is Asn119; this metal ion is also associated with five water molecules to complete an octahedral coordination complex. These water molecules are very well ordered and there is no evidence of rotational disorder or motion. Glu127 and His89 are located nearby and each is hydrogen bonded to water molecules in the coordination sphere. Asp86 is not chelated to the magnesium or its surrounding water molecules. Results of kinetics and site-specific mutagenesis experiments suggest that this metal-water cluster contains the catalytic metal ion of this enzyme. All residues which hydrogen bond to the water molecules that coordinate the magnesium atom are conserved in nucleases homologous to Serratia endonuclease, suggesting that the water cluster is a conserved feature of this family of enzymes. We offer a detailed structural comparison to one other nuclease, the homing endonuclease I-PpoI, that has recently been shown, in spite of a lack of sequence homology, to share a similar active site geometry to Serratia endonuclease. Evidence from both of these structures suggests that the magnesium of Serratia nuclease participates in catalysis via an inner sphere mechanism.     

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.     

Strand-specific contacts and divalent metal ion regulate double-strand break formation by the GIY-YIG homing endonuclease I-BmoI

GIY-YIG homing endonucleases are modular enzymes consisting of a well-defined N-terminal catalytic domain connected to a variable C-terminal DNA-binding domain. Previous studies have revealed that the role of the DNA-binding domain is to recognize and bind intronless DNA substrate, positioning the N-terminal catalytic domain such that it is poised to generate a staggered double-strand break by an unknown mechanism. Interactions of the N-terminal catalytic domain with intronless substrate are therefore a critical step in the reaction pathway but have been difficult to define. Here, we have taken advantage of the reduced activity of I-BmoI, an isoschizomer of the well-studied bacteriophage T4 homing endonuclease I-TevI, to examine double-strand break formation by I-BmoI. We present evidence demonstrating that I-BmoI generates a double-strand break by two sequential but chemically independent nicking reactions where divalent metal ion is a limiting factor in top-strand nicking. We also show by in-gel footprinting that contacts by the I-BmoI catalytic domain induce significant minor groove DNA distortions that occur independently of bottom-strand nicking. Bottom-strand contacts are critical for accurate top-strand nicking, whereas top-strand contacts have little influence on the accuracy of bottom-strand nicking. We discuss our results in the context of current models of GIY-YIG endonuclease function, with emphasis on the role of divalent metal ion and strand-specific contacts in regulating the activity of a single active site to generate a staggered double-strand break.     

Crystal structures of type II restriction endonuclease EcoO109I and its complex with cognate DNA

EcoO109I is a type II restriction endonuclease that recognizes the DNA sequence of RGGNCCY. Here we describe the crystal structures of EcoO109I and its complex with DNA. A comparison of the two structures shows that the catalytic domain moves drastically to capture the DNA. One metal ion and two water molecules are observed near the active site of the DNA complex. The metal ion is a Lewis acid that stabilizes the pentavalent phosphorus atom in the transition state. One water molecule, activated by Lys-126, attacks the phosphorus atom in an S(N)2 mechanism, whereas the other water interacts with the 3'-leaving oxygen to donate a proton to the oxygen. EcoO109I is similar to EcoRI family enzymes in terms of its DNA cleavage pattern and folding topology of the common motif in the catalytic domain, but it differs in the manner of DNA recognition. Our findings propose a novel classification of the type II restriction endonucleases and lead to the suggestion that EcoO109I represents a new subclass of the EcoRI family.     

Acidic residues and a predicted,highly conserved α‐helix are critical for the endonuclease/strand separation functions of bacteriophage λ’s TerL

Complementation, endonuclease, strand separation, and packaging assays using mutant TerL^λ’s, coupled with bioinformatic information and modeling of its endonuclease, identified five residues, D401, E408, D465, E563, and E586, as critical acidic residues of TerL^λ’s endonuclease. Studies of phage and viral TerL nucleases indicate acidic residues participate in metal ion‐binding, part of a two‐ion metal catalysis mechanism, where metal ion A activates a water for DNA backbone hydrolysis. Modeling places D401, D465, and E586 in locations analogous to those of the metal‐binding residues of many phage and viral TerLs. Our work leads to a model of TerL^λ’s endonuclease domain where at least three acidic residues from a ~185 residue segment (D401 to E586) are near each other in the structure, forming the endonuclease catalytic center at cosN, the nicking site. DNA interactions required to bring the rotationally symmetric cosN precisely to the catalytic center are proposed to rely on an ~60 residue region that includes a conserved α‐helix for dimerization. Metal ion A, positioned by TerL^λ’s acidic D401 and E586, would be placed at cosN for water activation, ensuring high accuracy for DNA backbone hydrolysis.     

A conserved tyrosine residue aids ternary complex formation,but not catalysis,in phage T5 flap endonuclease

The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose primary role appears to be chelation of essential divalent cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved tyrosine residue lies at the base of the archway in phage T5 flap endonuclease. This residue is conserved in the structures of all flap endonucleases analysed to date. We mutated the tyrosine 82 codon in the cloned T5 5' nuclease to one encoding phenylalanine. Detailed analysis of the purified Y82F protein revealed only a modest (3.5-fold) decrease in binding affinity for DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific endonuclease and exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type protein. The pH dependence of the kinetic parameters for the Y82F enzyme-catalysed reaction was bell-shaped as for the wild-type protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large (approximately 300-fold) defect in K(M). These results imply that this conserved tyrosine plays a key role in ternary complex formation (protein-DNA-metal ion), a prerequisite for catalysis.     

The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA

The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.     

New insight into the recognition of branched DNA structure by junction-resolving enzymes

Junction-resolving enzymes are nucleases that exhibit structural selectivity for the four-way (Holliday) junction in DNA. In general, these enzymes both recognize and distort the structure of the junction. New insight into the molecular recognition processes has been provided by two recent co-crystal structures of resolving enzymes bound to four-way DNA junctions in highly contrasting ways. T4 endonuclease VII binds the junction in an open conformation to an approximately flat binding surface whereas T7 endonuclease I envelops the junction, which retains a much more three-dimensional structure. Both proteins make contacts with the DNA backbone over an extensive area in order to generate structural specificity. The comparison highlights the versatility of Holliday junction resolution, and extracts some general principles of recognition.     

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

Background

The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally.

Results

Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme.

Conclusion

Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.     

The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases

The HNH motif is a small nucleic acid binding and cleavage module, widespread in metal finger endonucleases in all life kingdoms. Here we studied a non-specific endonuclease, the nuclease domain of ColE7 (N-ColE7), to decipher the role of the conserved asparagine and histidine residues in the HNH motif. We found, using fluorescence resonance energy transfer (FRET) assays, that the DNA hydrolysis activity of H545 N-ColE7 mutants was completely abolished while activities of N560 and H573 mutants varied from 6.9% to 83.2% of the wild-type activity. The crystal structures of three N-ColE7 mutants in complex with the inhibitor Im7, N560A-Im7, N560D-Im7 and H573A-Im7, were determined at a resolution of 1.9 A to 2.2 A. H573 is responsible for metal ion binding in the wild-type protein, as the zinc ion is still partially associated in the structure of H573A, suggesting that H573 plays a supportive role in metal binding. Both N560A and N560D contain a disordered loop in the HNH motif due to the disruption of the hydrogen bond network surrounding the side-chain of residue 560, and as a result, the imidazole ring of the general base residue H545 is tilted slightly and the scissile phosphate is shifted, leading to the large reductions in hydrolysis activities. These results suggest that the highly conserved asparagine in the HNH motif, in general, plays a structural role in constraining the loop in the metal finger structure and keeping the general base histidine and scissile phosphate in the correct position for DNA hydrolysis.     

Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.     

Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5′-CCGC/GG-3′ (“/” marks the cleavage position) and cuts it to generate 3′-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.     

The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits <5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k(cat)) and site of incision, and is as efficient (k(cat)/K(m)) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.     

Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease

Restriction endonuclease Bse634I recognizes and cleaves the degenerate DNA sequence 5'-R/CCGGY-3' (R stands for A or G; Y for T or C, '/' indicates a cleavage position). Here, we report the crystal structures of the Bse634I R226A mutant complexed with cognate oligoduplexes containing ACCGGT and GCCGGC sites, respectively. In the crystal, all potential H-bond donor and acceptor atoms on the base edges of the conserved CCGG core are engaged in the interactions with Bse634I amino acid residues located on the α6 helix. In contrast, direct contacts between the protein and outer base pairs are limited to van der Waals contact between the purine nucleobase and Pro203 residue in the major groove and a single H-bond between the O2 atom of the outer pyrimidine and the side chain of the Asn73 residue in the minor groove. Structural data coupled with biochemical experiments suggest that both van der Waals interactions and indirect readout contribute to the discrimination of the degenerate base pair by Bse634I. Structure comparison between related enzymes Bse634I (R/CCGGY), NgoMIV (G/CCGGC) and SgrAI (CR/CCGGYG) reveals how different specificities are achieved within a conserved structural core.     

Cruciform-resolvase interactions in supercoiled DNA

T4 endonuclease VII, which cleaves Holliday-like junctions in DNA, specifically cleaves short inverted repeats in supercoiled plasmids. These sequences are subject to site-specific cleavage by single-strand-specific nucleases, and cruciform formation has been suggested as an explanation for this observation. This proposal is greatly strengthened by the present data, since a formal analogy between cruciform structures and Holliday junctions exists. Resolution of a variety of unrelated cruciform sequences demonstrates that the cleavage process results in a linear molecule with hairpin ends and single ligatable nicks at positions corresponding to the stem-base of the cruciform. In two examples mapped in detail, the cleavages are exclusively introduced at two or three nucleotides from the end of the symmetric sequence at the 5' side on each strand. These studies demonstrate the potential of endonuclease VII as a probe of cruciform structure and the utility of short cruciform structures as Holliday junction models.