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.     

X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture

Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches. We have determined the crystal structures of the Ca2+-bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 A, respectively. The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif. The unique monomer fold, almost completely lacking beta-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction-resolving enzymes, including the Escherichia coli RuvC and lambda integrase-type recombinases. The S-shaped dimer has two 'binding bays' separated by approximately 25 A which are lined by positively charged residues and contain near their base residues known to be essential for activity. These include Asp40 and Asn62, which function as ligands for the bound calcium ions. A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations. A model for the complex with a four-way DNA junction is presented.     

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.     

T4 endonuclease VII cleaves holliday structures

T4 endonuclease VII cleaves Holliday structures in vitro by cutting two strands of the same polarity at or near the branch point. The two unbranched duplexes produced by cleavage each contain a strand break that can be sealed by DNA ligase. This suggests that the cut sites are at the same position in the nucleotide sequence in each strand. The joint action of endonuclease VII and DNA ligase can therefore resolve Holliday structures into genetically sensible products. These observations account for the role of endonuclease VII in the DNA metabolism of phage T4, and provide the first example of an enzyme that acts specifically on branch points in duplex DNA.     

Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.     

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.     

Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding complex between T7 endonuclease I and a synthetic Holliday junction analog has been probed with hydroxyl radicals. The results indicate that the nuclease binds all four strands about the junction point.     

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.     

Large-scale preparation of T4 endonuclease VII from over-expressing bacteria

Endonuclease VII is the product of gene 49 of phage T4 and was the first enzyme shown to resolve Holliday structures in vitro [Mizuuchi, K. et al. (1982) Cell 29, 357-365]. Low amounts of the enzyme were originally purified from phage-infected cells [Kemper, B. & Garabett, M. (1981) Eur. J. Biochem. 115, 123-131]. We now report a purification procedure for milligram amounts of cloned endonuclease VII expressed in Escherichia coli with gene 49 under the control of a temperature-inducible promoter on a plasmid system [Tomaschewski, J. (1988) PhD Thesis, University of Bochum, FRG]. The protein was purified 500-fold from crude extracts in five steps with a recovery of 15%. The steps include (a) poly(ethyleneglycol)/dextran two-phase separation; (b) DEAE-cellulose; (c) single-stranded DNA-agarose; (d) Mono-Q and (e) Mono-S chromatography. The final protein was more than 98% pure as estimated from SDS/PAGE analysis. The protein has an apparent molecular mass of 17.8 kDa on SDS-containing polyacrylamide gels and 36 kDa when determined by gel filtration or sedimentation through sucrose gradients in the presence of high salt (600 mM NaCl). In the absence of additional salt, the enzyme has a tendency to aggregate and products of molecular masses differing in steps of about 18 kDa appear on SDS-containing polyacrylamide gels.     

Thermodynamic properties of an intramolecular DNA four-way junction

We have investigated the thermodynamic properties of two homologous DNA four-way junctions, J4 and J4M, based on 46-mer linear DNA molecules. J4 and J4M have the same base sequence with the only difference that the latter contains an uncharged methylene-acetal linkage, -O3'-CH2-O5', instead of the phosphodiester linkage, -O3'-PO2-O5'-, between the residues T18 and C19. The comparison of the thermal unfolding of the J4 junction and J4M junction serves to investigate the effect of the uncharged methylene-acetal linkage on the stability of the junction. Our analysis is based on CD, UV absorbance spectroscopy, DSC, and chemical footprinting. The aim is to characterize in detail the structure and stability of the junctions. As demonstrated before by NMR, in the presence of 5 mM MgCl2 +/- 50 mM NaCl, both J4 and J4M form a complete four-way junction. This is now evidenced by protection from OsO4 cleavage (chemical footprinting). We can assume that full base pairing occurs throughout the arms even at the center of the junction. CD spectra suggest that the helices within the junctions adopt the regular B-DNA conformation. Almost identical melting temperatures and unfolding enthalpies are obtained for J4 and J4M both by UV and DSC. Furthermore, the Van't Hoff enthalpy (DeltaHVH) derived from UV melting equals the calorimetric enthalpy (DeltaHcal), which means that the melting process of the structures proceeds in a two-state manner. All results taken together support the conclusion that there are no major conformational and energetic differences between J4 and J4M. The inclusion of the uncharged methylene-acetal group into the junction has no effect on its stability.     

Structural recognition between a four-way DNA junction and a resolving enzyme

Resolving enzymes bind highly selectively to four-way DNA junctions, but the mechanism of this structural specificity is poorly understood. In this study, we have explored the role of interactions between the dimeric enzyme and the helical arms of the junction, using junctions with either shortened arms, or circular permutation of arms. We find that DNA-protein contacts in the arms containing the 5' ends of the continuous strands are very important, conferring a significant level of sequence discrimination upon both the choice of conformer and the order of strand cleavage. We have exploited these properties to obtain hydroxyl radical footprinting data on endonuclease I-junction complexes that are not complicated by the presence of alternative conformers, with results that are in good agreement with the arm permutation and shortening experiments. Substitution of phosphate groups at the center of the junction reveals the importance of electrostatic interactions at the point of strand exchange in the complex. Our data show that the form of the complex between endonuclease I and a DNA junction depends on the core of the junction and on interactions with the first six base-pairs of the arms containing the 5' ends of the continuous strands.     

Gel electrophoretic analysis of the geometry of a DNA four-way junction

Branched DNA molecules (Holliday structures) are believed to be key intermediates in the process of homologous genetic recombination. However, despite the importance of such structures, their transient nature makes it difficult to analyze their physical properties. In an effort to evaluate several models for the geometry of such branched molecules, a stable, synthetic DNA four-way junction has been constructed. The geometry of the synthetic junction has been probed by gel electrophoresis, utilizing the fact that bent DNA molecules demonstrate reduced mobilities on polyacrylamide gels to an extent that varies with the degree of the bend angle. From the synthetic four-way junction, we have produced a set of molecules in which all combinations of two junction arms have been extended by 105 base-pairs. The electrophoretic mobilities of the extended junctions differ in a manner which indicates that the junction is not a completely flexible structure; nor is it tetrahedral or planar-tetragonal. Instead, the four strands that comprise the DNA four-way junction are structurally non-equivalent. The significance of these observations with regard to previous models for four-way junction geometry is discussed.     

The stereochemistry of a four-way DNA junction: a theoretical study.

The stereochemical conformation of the four-way helical junction in DNA (the Holliday junction; the postulated central intermediate of genetic recombination) has been analysed, using molecular mechanical computer modelling. A version of the AMBER program package was employed, that had been modified to include the influence of counterions and a global optimisation procedure. Starting from an extended planar structure, the conformation was varied in order to minimise the energy, and we discuss three structures obtained by this procedure. One structure is closely related to a square-planar cross, in which there is no stacking interaction between the four double helical stems. This structure is probably closely similar to that observed experimentally in the absence of cations. The remaining two structures are based on related, yet distinct, conformations, in which there is pairwise coaxial stacking of neighbouring stems. In these structures, the four DNA stems adopt the form of two quasi-continuous helices, in which base stacking is very similar to that found in standard B-DNA geometry. The two stacked helices so formed are not aligned parallel to each other, but subtend an angle of approximately 60 degrees. The strands that exchange between one stacked helix and the other are disposed about the smaller angle of the cross (i.e. 60 degrees rather than 120 degrees), generating an approximately antiparallel alignment of DNA sequences. This structure is precisely the stacked X-structure proposed on the basis of experimental data. The calculations indicate distortions from standard B-DNA conformation that are required to adopt the stacked X-structure; a widening of the minor groove at the junction, and reorientation of the central phosphate groups of the exchanging strands. An important feature of the stacked X-structure is that it presents two structurally distinct sides. These may be recognised differently by enzymes, providing a rationalisation for the points of cleavage by Holliday resolvases.     

Bypass of a primase requirement for bacteriophage T4 DNA replication in vivo by a recombination enzyme, endonuclease VII.

A primase, the product of phage T4 gene 61, is required to initiate synthesis of Okazaki pieces and to allow bidirectional replication from several T4 origins. However, primase-defective T4 gene 61 mutants are viable. In these mutants, leading-strand DNA synthesis starts at the same time as in wild type infections, but, in contrast to wild type, initiation is unidirectional and the first replicative intermediates are large displacement loops. Rapid double-strand DNA replication occurs later after infection, generating multiple branched concatemers, which are cut and packaged into viable progeny particles, as in wild-type T4. Evidence is presented that this late double-strand DNA replication requires functional endonuclease VII (endo VII), the product of the T4 gene 49. We propose that endo VII can provide a backup mechanism when primase is defective, because it cuts recombinational junctions, generating 3' ends. These ends can prime DNA synthesis to copy the DNA strands that had been displaced during the initial origin-dependent replication. We explain the DNA-delay phenotype and the commonly observed temperature dependence of DNA replication in primase-deficient gene 61 mutants as a consequence of temperature-dependent translational control of gene 49 expression. In the presence or absence of functional primase endo VII is essential for correct packaging of DNA. The powerful selection that keeps the function of endo VII and expression of its gene at levels that are optimal for T4 development determines both the efficiency and the limitations of the bypass mechanism.     

Construction of a DNA four-way junction: Design and NMR spectroscopy

In 1964 Holliday postulated the formation of cruciform structures (four-way junctions) in duplex DNA as intermediate in genetic recombination. Since then, many biochemical and biophysical investigations were directed at solving questions concerning structural details of stable four-way junctions. Thus far, NMR spectroscopy played a minor part in these investigations on account of the minimum size of the molecule (expressed as the number of nucleotide residues) that was thought necessary to produce a stable cruciform structure. Indeed, the smallest four-way junction studied thus far by NMR methods was built from four separate DNA strands, each containing 16 nucleotides, a total of 64. Obviously, with such a large structure one runs into assignment problems. We considered the possibility of constructing a stable four-way junction from a single strand of DNA. The underlying idea was to make use of our detailed knowledge of the building principles of stable minihairpin loops. These loops, containing only two nucleotides to bridge the gap between antiparallel strands, are maximally stable in DNA sequences like 5-d(-C-TT-G-), where C and G form a normal Watson-Crick base pair and the two T residues cross the minor groove to form the minihairpin loop. Three of such miniloops could in principle cap three arms of the cruciform. The fourth arm would have an open end. The problem to be solved is to find the minimum length that is required to insure stability of the three closed arms and of the fourth open arm. We were successful with a structure that has three short stems (four base pairs each) and an open-end stem consisting of eight base pairs, a total of 46 residues. NMR experiments, carried out on this molecule in the presence of Mg²⁺, showed details of folding which have never been observed before.     

Interaction of T4 endonuclease V with DNA: importance of the flexible loop regions in protein-DNA interaction

T4 endonuclease V (T4 endo V), a thymine dimer-specific DNA repair enzyme, and its interaction with DNA were investigated by nuclear magnetic resonance (NMR) spectroscopy. Backbone resonance assignment, chemical shift mapping, and 15N relaxation measurements were employed to the free and DNA-bound enzymes. The secondary structure and the tertiary fold of T4 endo V in solution were consistent with those from the crystallographic study. The backbone 1H and 15N chemical shift perturbation upon the addition of DNA without a lesion revealed that the residues including Arg3, Arg22-Arg26, Lys45-Phe60, and Lys86-Thr88 participate in DNA binding. However, when DNA with a lesion was added to the enzyme and concomitantly the catalytic reaction was completed, the resonances of Arg22, Glu23, and Arg26, which constitute the catalytic active site, and the resonance of Thr88, were perturbed in a different manner. The region around Lys45-Ser47 was found to be involved in DNA binding, which have not been reported elsewhere. The backbone relaxation measurements of the free and DNA-bound enzymes indicated that two loop regions, Lys45-Phe60 and Lys86-Asp92, show the high degree of backbone flexibility. These results imply that two flexible loop regions may play an important role in DNA binding and in scanning along DNA duplex to search the thymine dimer sites in UV-damaged DNA.     

Initiation of heteroduplex-loop repair by T4-encoded endonuclease VII in vitro.

Heteroduplex DNAs with single-stranded loops of 51 nt or 8 nt were constructed in vitro and used in reactions with purified endonuclease VII (endo VII) from phage T4. The enzyme makes double-strand breaks by introducing pairs of staggered nicks flanking the loops. Regardless of loop-size the nicking sites map exclusively at the 3' side of the loop in the looping strand and at the 3' side of the base of the loop in the non-looping strand. The number of potential cleavage sites is small (less than 5) and their distribution depends on DNA sequence. The two closest staggered nicks are 4 bp apart, 2 bp on either side of the loop. Nicking always occurs in the double-stranded part of the molecules; the single-stranded loops are not attacked by endo VII. The nicks are introduced in a stepwise fashion and selection of the strand for the first nick depends on the sequence of 31 base pairs flanking the loops.     

Dynamics of the T4 bacteriophage DNA packasome motor: endonuclease VII resolvase release of arrested Y-DNA substrates

Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.     

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.