The complex between a four-way DNA junction and T7 endonuclease I

The junction-resolving enzyme endonuclease I is selective for the structure of the DNA four-way (Holliday) junction. The enzyme binds to a four-way junction in two possible orientations, with a 4:1 ratio, opening the DNA structure at the centre and changing the global structure into a 90 degrees cross of approximately coaxial helices. The nuclease cleaves the continuous strands of the junction in each orientation. Binding leads to pronounced regions of protection of the DNA against hydroxyl radical attack. Using all this information together with the known structure of the enzyme and the structure of the BglI-DNA complex, we have constructed a model of the complex of endonuclease I and a DNA junction. This shows how the enzyme is selective for the structure of a four-way junction, such that both continuous strands can be accommodated into the two active sites so that a productive resolution event is possible.     

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.     

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.     

T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein.

T7 endonuclease I is known to bind and cleave four-way junctions in DNA. Since these junctions serve as analogues of Holliday junctions that arise during genetic recombination, we have investigated the action of T7 endonuclease I on recombination intermediates containing Holliday junctions. We find that addition of T7 endonuclease I to strand exchange reactions catalysed by RecA protein of Escherichia coli leads to the formation of duplex products that correspond to 'patch' and 'splice' type recombinants. Resolution of the recombination intermediates occurs by the introduction of nicks at the site of the Holliday junction. The recombinant molecules contain 5'-phosphate and 3'-hydroxyl termini which may be ligated to restore the integrity of the DNA.     

Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA.

A survey of restriction endonucleases having different cleavage specificities has identified 10 that do not cut wild-type bacteriophage T7 DNA, 11 that cut at six or fewer sites, four that cut at 18 to 45 sites, and 12 that cut at more than 50 sites. All the cleavage sites for the 13 enzymes that cut at 26 or fewer sites have been mapped. Cleavage sites for each of the 10 enzymes that do not cut T7 DNA would be expected to occur an average of 9 to 10 times in a random nucleotide sequence the length of T7 DNA. A possible explanation for the lack of any cleavage sites for these enzymes might be that T7 encounters enzymes having these specificities in natural hosts, and that the sites have been eliminated from T7 DNA by natural selection. Five restriction endonucleases were found to cut within the terminal repetition of T7 DNA; one of these, KpnI, cuts at only three additional sites in the T7 DNA molecule. The length of the terminal repetition was estimated by two independent means to be approximately 155 to 160 base-pairs.     

The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I

Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.     

Brownian-dynamics simulations of metal-ion binding to four-way junctions

Four-way junctions (4Hs) are important intermediates in DNA rearrangements such as genetic recombination. Under the influence of multivalent cations these molecules undergo a conformational change, from an extended planar form to a quasi-continuous stacked X-structure. Recently, a number of X-ray structures and a nuclear magnetic resonance (NMR) structure of 4Hs have been reported and in three of these the position of multivalent cations is revealed. These structures belong to two main families, characterized by the angle between the two co-axial stacked helices, which is either around +40 to +55° or around –70 to –80°. To investigate the role of metal-ion binding on the conformation of folded 4Hs we performed Brownian-dynamics simulations on the set of available structures. The simulations confirm the proposed metal-ion binding sites in the NMR structure and in one of the X-ray structures. Furthermore, the calculations suggest positions for metal-ion binding in the other X-ray structures. The results show a striking dependence of the ion density on the helical environment (B-helix or A-helix) and the structural family.     

Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.     

Interaction of a four-way junction in DNA with T4 endonuclease VII

The binding of a synthetic four-way junction in DNA by T4 endonuclease VII has been studied using gel retardation and footprint analysis. Two specific protein-DNA complexes have been observed, but only one is stable in the presence of moderate concentrations of salt. The footprint of T4 endonuclease VII in the salt-resistant complex has been probed using hydroxyl radicals generated by the reaction of iron(II)/EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern indicates protection of approximately 5 residues in two strands that are diametrically opposed across the junction point.     

Gene 3 endonuclease of bacteriophage T7 resolves conformationally branched structures in double-stranded DNA

Gene 3 endonuclease of bacteriophage T7 has been expressed from the cloned gene, purified, and characterized as to its activity on different DNA substrates. Besides its known strong preference for cutting single-stranded DNA rather than double-stranded DNA, the enzyme has a strong preference for cutting conformationally branched structures in double-stranded DNA, either X or Y-shaped branches. Three types of branched DNA substrates were used: relaxed circular DNAs containing large cruciform structures (a model for Holliday structures, presumed intermediates in genetic recombination); X-shaped molecules having a limited potential for branch migration, made from the cloned phage and bacterial arms of the lambda attachment site; and Y-shaped molecules, made by hybridizing molecules homologous except for a 2 X 21 base-pair palindrome in one of them. Gene 3 endonuclease cuts two opposing strands at or near the branchpoint to resolve these substrates into linear molecules, and does not cut the potentially single-stranded tips of the stem-and-loop structure generated from the palindrome. The position of the cleavage points on the equivalent arm of two X-shaped molecules, constructed from wild-type and mutant lambda attachment sites, show that the enzyme can cut at several different sites within or slightly 5' of the limited region of branch migration. The various activities of gene 3 endonuclease are consistent with the known role of this enzyme in genetic recombination, in maturation and packaging of T7 DNA, and in degradation of host DNA, and suggest that the enzyme recognizes a specific structural feature in DNA. Its cleavage specificity, ready availability, and ability to act at physiological pH and ionic conditions may make gene 3 endonuclease useful as a probe for specific DNA structures or for binding of proteins that alter DNA structure.     

Specificity from the synapsis of DNA elements by the Sfi I endonuclease.

The synapsis of DNA sites is a prerequisite for the reactions of many proteins that act at specific DNA sequences. The requirement for synapsis was investigated by analysing the reactions of Sfi I, a tetrameric restriction enzyme that cleaves DNA only after interacting with two recognition sites. In the presence of Mg2+, oligonucleotide duplexes with the cognate recognition sequence were cleaved rapidly, with cooperative kinetics, while non-cognate duplexes were not cleaved. In the absence of Mg2+, the primary complex formed by Sfi I with cognate DNA contained two duplexes synapsed by the tetramer: a secondary complex containing one duplex was seen only at elevated Sfi I concentrations. In contrast, the principal complex with non-cognate DNA contained one duplex bound to Sfi I. Pairs of non-cognate duplexes, or one cognate and one non-cognate duplex, generally failed to form synaptic complexes. On adding Mg2+to complexes with cognate DNA, cleavage occurred much more rapidly in the synaptic complex than in the secondary complex. DNA synapsis thus acts to enhance the specificity of Sfi I for its recognition sequence, by demanding two cognate sites for a catalytically active complex and by excluding non-cognate sites from the synaptic complex.     

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.     

Resolution of branched DNA substrates by T7 endonuclease I and its inhibition

Endonuclease I is a multipurpose enzyme implicated in the breakdown of host DNA, packaging of phage DNA, and recombination during the lytic cycle of bacteriophage T7. We investigate here some aspects of the substrate requirements for its activity in resolving branched intermediates similar to Holliday junctions (Holliday, R. (1964) Genet. Res. 5, 282-304) that arise in recombination. The enzyme is able to resolve branched substrates containing very short duplex arms: 4 base pairs suffice. It cleaves 5' to the branch, with a distinct preference for the non-crossover strands in Holliday-like model junctions. Ligands that interact strongly with the branch site can inhibit the enzyme, with KI values in the 10-50 microM range.     

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.     

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.     

Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA

The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA. All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C. C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 205-209). Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence. Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter. The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate. Of the eight common nucleoside triphosphates, dTTP promotes optimal binding. The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed. Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min. In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

Defective DNA injection by alkylated and nonalkylated bacteriophage T7.

DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA. Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells. This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome. An essentially identical injection defect was observed when alkylated phage infected E. coli mutant cells unable to repair 3-methyladenine. Furthermore, untreated phage particles were discovered to be naturally injection-defective. Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all. In the presence of rifampicin, untreated phages injected only segments from the left end of the genome. These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome. The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E. coli cells. Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.