The active site of the junction-resolving enzyme T7 endonuclease I

Endonuclease I is a junction-resolving enzyme encoded by bacteriophage T7, that selectively binds and cleaves four-way DNA junctions. We have recently solved the structure of this dimeric enzyme at atomic resolution, and identified the probable catalytic residues. The putative active site comprises the side-chains of three acidic amino acids (Glu20, Asp55 and Glu65) together with a lysine residue (Lys67), and shares strong similarities with a number of type II restriction enzymes. However, it differs from a typical restriction enzyme as the proposed catalytic residues in both active sites are contributed by both polypeptides of the dimer. Mutagenesis experiments confirm the importance of all the proposed active site residues. We have carried out in vitro complementation experiments using heterodimers formed from mutants in different active site residues, showing that Glu20 is located on a different monomer from the remaining amino acid residues comprising the active site. These experiments confirm that the helix-exchanged architecture of the enzyme creates a mixed active site in solution. Such a composite active site structure should result in unilateral cleavage by the complemented heterodimer; this has been confirmed by the use of a cruciform substrate. Based upon analogy with closely similar restriction enzyme active sites and our mutagenesis experiments, we propose a two-metal ion mechanism for the hydrolytic cleavage of DNA junctions.     

Mechanistic aspects of the DNA junction-resolving enzyme T7 endonuclease I

The chemical mechanism of phosphodiester bond hydrolysis catalyzed by a junction-resolving enzyme has been investigated. Endonuclease I of phage T7 is a member of the nuclease superfamily of proteins that include many restriction enzymes, and the structure of the active site is very similar to that of BglI in particular. It contains three acidic amino acids that coordinate two divalent metal ions. Using mass spectrometry we have shown that endonuclease I catalyzes the breakage of the P-O3' bond, in common with restriction enzymes. We have found that the pH dependence of the hydrolysis reaction is log-linear, with a gradient of 0.9. Substitution of the scissile phosphate by an electrically neutral methylphosphonate significantly impairs the rate of bond cleavage. However, the introduction of chirally pure methylphosphonate groups shows that the effect of substitution of the proS oxygen atom is much greater than that for the proR. This is consistent with our current model of the structure of the DNA bound in the active site of endonuclease I, where the proS oxygen atom is coordinated directly to both metal ions as it is in BglI. The activity is also very sensitive to repositioning of the carboxylate groups of Asp 55 and Glu 65 in the active site, although some restoration of activity in endonuclease I E65D was observed in the presence of Mn2+ ions. A mechanism of hydrolysis consistent with all of these data is proposed.     

Metal ion binding in the active site of the junction-resolving enzyme T7 endonuclease I in the presence and in the absence of DNA

Endonuclease I of bacteriophage T7 is a DNA junction-resolving enzyme. We have previously used crystallography to demonstrate the binding of two manganese ions into the active site that is formed by three carboxylate (Glu 20, Asp 55 and Glu 65) and a lysine residue (Lys 67). Endonuclease I is active in the presence of magnesium, manganese, iron (II) and cobalt (II) ions, weakly active in the presence of nickel, copper (II) and zinc ions, and completely inactive in the presence of calcium ions. However, using calorimetry, we have observed the binding of two calcium ions to the free enzyme in a manner very similar to the binding of manganese ions. In the presence of iron (II) ions, we have obtained a cleavage of the continuous strands of a junction bound by endonuclease I, at sites close to (but not identical with) enzyme-induced hydrolysis. The results suggest that this arises from attack by locally generated hydroxyl radicals, arising from iron (II) ions bound into the active site. This therefore provides an indirect way of examining metal ion binding in the enzyme-junction complex. Ion binding in free protein (by calorimetry) and the enzyme-junction complex (iron-induced cleavage) have been studied in series of active-site mutants. Both confirm the importance of the three carboxylate ligands, and the lack of a requirement for Lys67 for the ion binding. Calorimetry points to particularly critical role of Asp55, as mutation completely abolishes all binding of both manganese and calcium ions.     

A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease

A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure.     

Analysis of conformational changes in the DNA junction-resolving enzyme T7 endonuclease I on binding a four-way junction using EPR

The four-way (Holliday) DNA junction is the central intermediate in homologous recombination. It is ultimately resolved into two nicked-duplex species by the action of a junction-resolving enzyme. These enzymes are highly selective for the structure of branched DNA, yet as a class these proteins impose significant distortion on their target junctions. Bacteriophage T7 endonuclease I selectively binds and cleaves DNA four-way junctions. The protein is an extremely stable dimer, comprising two globular domains joined by a β-strand bridge with each active site including amino acids from both polypeptides. The crystal structure of endonuclease I has been solved both as free protein and in complex with a DNA junction, showing that the protein, as well as the junction, becomes distorted on binding. We have therefore used site-specific spin-labeling in conjunction with EPR distance measurements to analyze induced fit in the binding of endonuclease I to a DNA four-way junction. The results support the change in protein structure as it binds to the junction. In addition, we have examined the structure of wild type and catalytically inactive mutants alone and in complex with DNA. We demonstrate the presence of hitherto undefined metastable conformational states within endonuclease I, showing how these states can be influenced by DNA-junction binding or mutations within the active sites. In addition, we demonstrate a previously unobserved instability in the N-terminal α1-helix upon active site mutation. These studies reveal that structural changes in both DNA and protein occur in the action of this junction-resolving enzyme.     

Near-simultaneous DNA cleavage by the subunits of the junction-resolving enzyme T4 endonuclease VII.

In common with a number of other DNA junction-resolving enzymes, endonuclease VII of bacteriophage T4 binds to a four-way DNA junction as a dimer, and cleaves two strands of the junction. We have used a supercoil-stabilized cruciform substrate to probe the simultaneity of cleavage at the two sites. Active endonuclease VII converts the supercoiled circular DNA directly into linear product, indicating that the two cleavage reactions must occur within the lifetime of the protein-junction complex. By contrast, a heterodimer of active enzyme and an inactive mutant endonuclease VII leads to the formation of nicked circular product, showing that the subunits operate fully independently.     

Catalytic and binding mutants of the junction-resolving enzyme endonuclease I of bacteriophage t7: role of acidic residues.

Endonuclease I is a 149 amino acid protein of bacteriophage T7 that is a Holliday junction-resolving enzyme, i.e. a four-way junction-selective nuclease. We have performed a systematic mutagenesis study of this protein, whereby all acidic amino acids have been individually replaced by other residues, mainly alanine. Out of 21 acidic residues, five (Glu20, Glu35, Glu65, Asp55 and Asp74) are essential. Replacement of these residues by other amino acids leads to a protein that is inactive in the cleavage of DNA junctions, but which nevertheless binds selectively to DNA junctions. The remaining 16 acidic residues can be replaced without loss of activity. The five critical amino acids are located within one section of the primary sequence. It is rather likely that their function is to bind one or more metal ions that coordinate the water molecule that brings about hydrolysis of the phosphodiester bond. We have also constructed a mutant of endonuclease I that lacks nine amino acids (six of which are arginine or lysine) at the C-terminus. Unlike the acidic point mutants, the C-terminal truncation is unable to bind to DNA junctions. It is therefore likely that the basic C-terminus is an important element in binding to the DNA junction.     

Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I

We have solved the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 A resolution using the multiwavelength anomalous dispersion (MAD) technique. Endonuclease I exhibits strong structural specificity for four-way DNA junctions. The structure shows that it forms a symmetric homodimer arranged in two well-separated domains. Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site. While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases. T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes.     

The mechanism of the amyloidogenic conversion of T7 endonuclease I

Amyloid fibrils are associated with a range of human disorders. Understanding the conversion of amyloidogenic proteins from their soluble forms to amyloid fibrils is critical for developing effective therapeutics. Previously we showed that T7 endonuclease I forms amyloid-like fibrils. Here we study the mechanism of the amyloidogenic conversion of T7 endonuclease I. We show that T7 endonuclease I forms fibrils at pH 6.8, but not at pH 6.0 or 8.0. The amyloidogenicity at pH 6.8 is not correlated with thermodynamic stability, unfolding cooperativity, or solubility. Thermal melting experiments at various pH values show that the protein has a distinctive thermal transition at pH 6.8. The transition at pH 6.8 has a lower transition temperature than the unfolding transitions observed at pH 6.0 and 8.0 and leads to a beta-rich conformation instead of an unfolded state. Electron microscopy shows that the thermal transition at pH 6.8 results in fibril formation. The thermal transition at pH 6.8 leads to a protein state that is not accessible at pH 6.0 or 8.0, showing that the existence of the amyloidogenic conformation of T7 endonuclease I depends sensitively on solution conditions. Therefore, we propose that fibrillizing proteins need to be "prepared" for fibrillization. Preparation may consist of amino acid replacements or changing solution conditions and may require retention of some aspects of native structure. In this model, some amyloid-enhancing mutations decrease protein stability, whereas others have little effect.     

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.     

A catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions

In efforts to understand the mechanisms of many nucleic acid enzymes, the first site-directed mutations are made at conserved acidic active residues. Almost without exception, the low or null activities of the resulting variants are attributed to the importance of the acidic residue(s) to the ligation of required metal ions. Using (25)Mg NMR spectroscopy as a direct probe of metal ion binding and the homodimeric PvuII restriction endonuclease as a model system, this interpretation is examined and clarified. Our results indicate that Mg(II) binds wild-type PvuII endonuclease in the absence of DNA with a K(d,app) of 1.9 mM. Hill analysis yields an n(H) coefficient of 1.4, a value consistent with the binding of more than one Mg(II) ion per monomer active site. Variable pH studies indicate that two ionizable groups are responsible for Mg(II) binding by wild-type PvuII endonuclease near physiological pH. The pK(a,app) for these ionizations is 6.7, a value which is unusual for acidic residues but consistent with data obtained for critical groups in MunI endonuclease and a number of other hydrolases. To assign residues critical to ligating Mg(II), binding measurements were performed on the low activity catalytic site mutants E68A and D58A. As expected, E68A binds Mg(II) ions very weakly (K(d,app) approximately 40 mM), implicating Glu68 as critical to Mg(II) binding. Interestingly, while D58A has only residual specific activity, it retains an affinity for Mg(II) with a K(d,app) of 3.6 mM and exhibits a Hill coefficient of 0.7. Moreover, in this variant, multiple ionizable groups with pK(a,app) of 7.2 are involved in Mg(II) binding, suggesting a shuffling of Mg(II) ligands in the active site. These data indicate that Asp58 is important for the critical positioning of metal ion(s) required for catalysis.     

CrystallographicB factor of critical residues at enzyme active site

Thirty-seven sets of crystallographic enzyme data were selected from Protein Data Bank (PDB, 1995). The average temperature factors (B) of the critical residues at the active site and the whole molecule of those enzymes were calculated respectively. The statistical results showed that the critical residues at the active site of most of the enzymes had lowerB factors than did the whole molecules, indicating that in the crystalline state the critical residues at the active site of the natural enzymes possess more stable conformation than do the whole molecules. The flexibility of the active site during the unfolding by denaturing was also discussed.     

Heteroduplex resolution using T7 endonuclease I in microbial community analyses

Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities. This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques. Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts. Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA. Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I. Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning. These samples were compared with replicates that did not receive the T7 endonuclease I treatment. Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones. Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened. The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.     

Functional residues at the active site of angiotensin converting enzyme.

A series of chemical modification reactions have been carried out with rabbit pulmonary angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) in order to identify amino acid residues essential for its catalytic activity. The enzyme is rapidly inactivated by nitration with tetranitromethane and by O-acetylation with N-acetylimidazole. Deacylation with hydroxylamine restores activity to the acetylated enzyme, while the inhibitor, β-phenylpropionyl-L-phenylalanine, protects against acetylimidazole inactivation. These results indicate the presence of functional tyrosyl residues at the active site of the enzyme. Reaction with butanedione decreases activity, an effect that is markedly enhanced by the presence of borate, indicating essential arginyl residues. In addition, activity is diminished by the carboxyl reagent, cyclohexylmorpholinoethyl carbodiimide. Thus, the three functional residues long known to be components of the active site of bovine carboxypeptidase A, tyrosyl, arginyl, and glutamyl, have counterparts in the angiotensin converting enzyme. The effects of pyridoxal phosphate and a number of other reagents demonstrate that the converting enzyme also contains an important lysyl residue.     

Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions

Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination. To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology. In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes. Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity. The major sites of cleavage were identified and found to occur within the boundaries of homology. T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues. However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences.