Endo VII Research Articles

F-CphI represents a new homing endonuclease family using the Endo VII catalytic motif

BackgroundThere are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Previously, we discovered a novel homing endonuclease F-CphI encoded by ORF177 of cyanophage S-PM2. F-CphI does not resemble any characterized homing endonucleases. Instead, the C-terminus of F-CphI aligns well with the N-terminal catalytic domain of a Holliday junction DNA resolvase, phage T4 endonuclease VII (Endo VII).ResultsA PSI-BLAST search resulted in a total of 313 Endo VII motif–containing sequences in sequenced genomes. Multiple sequence alignment showed that the catalytically important residues of T4 Endo VII were all well conserved in these proteins. Our site-directed mutagenesis studies further confirmed that the catalytically important residues of T4 Endo VII were also essential for F-CphI activity, and thus F-CphI might use a similar protein fold as Endo VII for DNA cleavage. A phylogenetic tree of the Endo VII motif–containing sequences showed that putative resolvases grouped into one clade while putative homing endonucleases and restriction endonucleases grouped into another clade.ConclusionsBased on the unique conserved residues, we proposed that F-CphI represents a new homing endonuclease family, which was named the DHHRN family. Our phylogenetic analysis could be used to predict the functions of many previously unknown proteins.

Endonuclease VII is a key component of the mismatch repair mechanism in bacteriophage T4

In previous papers we described an extra recombination mechanism in T4 phage, which contributed to general recombination only when particular mutations were used as geneticmarkers (high recombination or HR markers), whereas it was practically inactive towards other rIIB mutations (low recombination or LR markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombination heteroduplexes. We suggested that the first step in this pathway, recognition and incision of the mismatch, is performed by endonuclease VII (endo VII) encoded by the T4 gene 49. In the present paper, we tested this hypothesis in vivo. We used an experimental model system that combines site-specific double-strand breaks with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. We compared recombination of homoallelic HR and LR markers in the S17 and S17 E727 background (amber mutations in the uvsX and in the uvsX and 49 genes, respectively). In S17-crosses, the HR and LR markers retain their respective high-recombination and low-recombination behavior. In S17 E727-crosses, however, the HR and LR markers show no difference in the recombination frequency and both behave as LR markers. We conclude that endo VII is the enzyme that recognizes mismatches in recombinational heteroduplexes and performs their incision. This role for endo VII was suggested previously from biochemical studies, but this is its first in vivo demonstration.

Crystal structure of T4 endonuclease VII resolving a Holliday junction

Holliday proposed a four-way DNA junction as an intermediate in homologous recombination, and such Holliday junctions have since been identified as a central component in DNA recombination and repair. Phage T4 endonuclease VII (endo VII) was the first enzyme shown to resolve Holliday junctions into duplex DNAs by introducing symmetrical nicks in equivalent strands. Several Holliday junction resolvases have since been characterized, but an atomic structure of a resolvase complex with a Holliday junction remained elusive. Here we report the crystal structure of an inactive T4 endo VII(N62D) complexed with an immobile four-way junction with alternating arm lengths of 10 and 14 base pairs. The junction is a hybrid of the conventional square-planar and stacked-X conformation. Endo VII protrudes into the junction point from the minor groove side, opening it to a 14 A x 32 A parallelogram. This interaction interrupts the coaxial stacking, yet every base pair surrounding the junction remains intact. Additional interactions involve the positively charged protein and DNA phosphate backbones. Each scissile phosphate that is two base pairs from the crossover interacts with a Mg2+ ion in the active site. The similar overall shape and surface charge potential of the Holliday junction resolvases endo VII, RuvC, Ydc2, Hjc and RecU, despite having different folds, active site composition and DNA sequence preference, suggest a conserved binding mode for Holliday junctions.

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Two major pathways of recombination-dependent DNA replication, "join-copy" and "join-cut-copy," can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Analyses of spontaneous mutations of cloned gene 49 of phage T4

Holliday structure resolving enzyme endonuclease VII (endo VII) of phage T4 is highly toxic for E. coli when expressed outside of the phage infection environment. As a consequence, plasmids with a mutated gene 49, the gene which encodes for endo VII, can be easily isolated and characterised. We have isolated and characterised 400 survivors from independent transformations with a plasmid carrying gene 49 under the control of the T7 promoter. The majority had mutated gene 49 by IS 10 insertions which almost exclusively mapped to a distinct site. When this site was mutated other insertion sites were observed as well as an increase in other mutational events including large deletions. Neither of the observed insertion sites mapped matched the consensus IS 10 sequence completely. Additionally when the level of expression of gene 49 was altered the distribution of mutations was changed suggesting that other elements apart from the target sequence are necessary for determining IS 10 insertion. The expression of gene 49 in E. coli provides a particularly useful tool for the analysis of mutational events.

The X philes: structure-specific endonucleases that resolve Holliday junctions.

Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication. It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four-way branched intermediate commonly referred to as the Holliday junction. DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division. This resolution reaction is mediated by structure-specific endonucleases that catalyse dual-strand incision across the point of strand cross-over. Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands. Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination. Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA. They use a metal-binding pocket to co-ordinate an activated water molecule for phosphodiester bond hydrolysis. In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences. Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R). Recent studies have brought about a reappraisal of the origins of junction-specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core.

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.

Association of Holliday-structure Resolving Endonuclease VII with gp20 from the Packaging Machine of Phage T4

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacteriophage T4. It is a Holliday-structure resolvase (X-solvase) responsible for clearing branched replicative DNA prior to packaging. Consequently, mutations in gene 49 are unable to fill heads to completion because unresolved branches stop translocation of DNA. A likely association of gp49 with heads or proheads, however, could not be shown in the past. We have investigated whether gp49 could be part of the transiently assembled packaging machine (the “packasome”) located at the base of proheads. Using purified proteins gpl6, gpl7 and gp20, which are constituents of the packasome, we found that gp49 binds tightly to gp20 and does not bind to gpl6 or gpl7. Quantification revealed that one dimer of gp49 binds one monomer of gp20. Notably, dimerisation of gp49 was an essential prerequisite for complex formation with gp20, and the dimerisation-deficient point mutation His-EVII-W87R showed only residual affinity to gp20. Furthermore, truncated peptides of gp49 deficient in dimer formation to various degrees were found to be impaired in binding to gp20. In contrast, the cleavage-deficient mutation EVII-N62D bound normally to gp20. The cruciform DNA (cf-DNA) resolving activity typical of endo VII is maintained in gp20-gp49 complexes. Furthermore, the complexes bind cf-DNA in the absence of Mg 2+ as demonstrated by electromobility shift assays. The binding of the complexes to cf-DNA occurs via gp49, since gp20 alone does not bind cf-DNA. In conclusion, these findings are consistent with a model in which gp49 is an integral part of the packaging machine of phage T4.

An apyrimidinic site kinks DNA and triggers incision by endonuclease VII of phage T4.

Apurinic/apyrimidinic lesions (AP-sites) occur frequently in DNA, generated by physically and chemically induced or spontaneous loss of bases. Repair mechanisms have evolved in organisms to deal efficiently with AP-sites by first incising the DNA at the lesion, followed by excision and resynthesis of the damaged strand. Here we report that endonuclease VII (endo VII) of phage T4, which was originally classified as a debranching and Holliday structure resolving enzyme, also recognizes AP-sites with high efficiency. The enzyme cleaves both strands of double-stranded DNA in a stepwise fashion a few nucleotides 3' of the lesion. In a search for a recognition signal shared by all known endo VII substrates, kinking of DNA has earlier been suggested as such a signal. In support of this hypothesis, we demonstrate here that AP-sites induce distinct kinks in synthetic oligonucleotides allowing efficient intramolecular ring closure by ligation.

Endonuclease VII has two DNA-binding sites each composed from one N- and one C-terminus provided by different subunits of the protein dimer.

Endonuclease VII (endo VII) is a Holliday structure-resolving enzyme of bacteriophage T4. Its activity depends on dimerization, DNA binding and hydrolysis of two phosphodiester bonds flanking the Holliday junction. We analysed the DNA-binding activity of truncated monomeric and covalently linked dimeric endo VII proteins. We show that both ends of endo VII are involved in DNA binding. In particular, the C-terminus of one subunit interacts with the N-terminus of the other subunit, constituting one DNA-binding site; the other two termini form the second binding site of the dimer. One binding site is sufficient to bind cruciform DNA. The concerted mechanism involving termini from different subunits ensures that only dimers bind to Holliday structures, thus providing two catalytic centres which introduce two cleavages in opposite strands. This is a precondition for precise resolution of Holliday structures.

Localization and characterization of the dimerization domain of Holliday structure resolving endonuclease VII of phage T4

Endonuclease VII (Endo VII) is a Holliday structure resolving enzyme of bacteriophage T4. Its nucleolytic activity depends on subactivities, which in order of execution are: (i) dimerization, (ii) binding to DNA, (iii) and cleavage of DNA. In an effort to assign these subfunctions to the primary sequence of the protein, a series of spontaneous point mutations deficient in DNA cleavage was isolated. Some of these mutations affected the dimerization of Endo VII. Compared with wild-type protein, which dimerizes completely in solution, more than 95% of one of the mutant proteins (W87R) remained in the monomeric state. Only the dimeric fraction of this protein bound to DNA. The dimerization domain of Endo VII was mapped by truncating the gene from both ends and analysing the dimerization ability of the purified peptides by crosslinking with glutaraldehyde. The dimerization domain was thus determined to reside between amino acid residues 55 and 105. Computer analyses predicted two α-helices (H2 and H3) in this section of the protein. As demonstrated by heterodimer formation, two copies of helix H3, but only one copy of helix H2, are required for dimerization. Helical wheel analyses revealed that both helices expose a hydrophobic face along their axes, suggesting that hydrophobic interaction between helices H3 mediate formation of Endo VII dimers, while helices H2 stabilize them.

Enzymatic mutation detection. Phosphate ions increase incision efficiency of endonuclease VII at a variety of damage sites in DNA

The ability of endonuclease VII (endo VII) to cleave at mispairings in double-stranded DNA has recently been used for enzymatic mutation detection (EMD) [R. Youil, B.W. Kemper, R.G.H. Cotton, Proc. Natl. Acad. Sci. USA 92 (1995) 87-91]. The method is based on mapping cleavages in heteroduplex DNAs obtained from mutant and wildtype sequences. Despite the capability of endo VII to cleave at all possible mispairings, relative cleavage efficiencies vary considerably for individual mismatches and may escape detection if located in an unfavorable sequence surrounding. We report here improved reaction conditions which can increase the selectivity of the enzyme for mismatches up to 500-fold, as demonstrated with a mutation in a 247 nt long fragment from exon 7 of human gene p53. The new conditions involve replacement of Tris/HCl buffer by phosphate buffer and change from pH 8.0 to 6.5. Various concentrations of phosphate ions should be tried in the assay to meet individual requirements of the substrate.

Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding

Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs.

Enzymatic mutation detection. Procedure for screening and mapping of mutations by immobilised endonuclease VII.

Endonuclease VII (endo VII) binds to non-pairing nucleotides in DNA. This served as the basis for the development of a mutation detection assay involving immobilised endo VII and heteroduplex DNAs made by hybridisation of mutant and wild type DNA. The use of microtiter plates allows screening of large numbers of samples. Localisation of mutations in positive samples can be done in the same assay in a second optional step.

Inhibition of holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY

Proteins UvsX, UvsY and Endonuclease VII (Endo VII) of bacteriophage T4 are required for DNA recombination, replication and repair. Endo VII is the product of gene 49 (gp49) and essential for resolution of branches from newly made DNA, prior to packaging into preformed heads. The ability of Endo VII to resolve Holliday structures in vitro suggested an in vivo function for the resolution of recombination intermediates, generated by UvsX and UvsY during the early infection cycle. Here we report results which contrast with this hypothesis. It is shown that the potent endonucleolytic activity of Endo VII with branched DNAs is inhibited in strand transfer reactions by the strand transferase UvsX, and more strongly by the accessory protein UvsY in vitro. The inhibitory effect of UvsX or UvsY is also seen in reactions with Endo VII using two synthetic cruciform DNAs and a C/C-mismatch containing substrate. Low concentrations of UvsY protein (12 ng or 0,76 pmol) were sufficient to reduce the cleavage efficiency of 30 units of Endo VII (about 16 fmol) to 50%. The inhibition is due to a direct protein-protein interaction between Endo VII, UvsX and UvsY as suggested by electrophoretic mobility shift assays (EMSAs). These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immobilized on a NHS-activated Sepharose matrix. This is the first identification of phage-encoded proteins which modulate the potent endonucleolytic activity of gp49 in vitro.

Identification of double holliday junctions as intermediates in meiotic recombination

During meiosis, branched DNA molecules containing information from both parental chromosomes occur in vivo at loci where meiosis-specific double-stranded breaks occur. We demonstrate here that these joint molecules are recombination intermediates: they contain single strands that have undergone exchange of information. Moreover, these joint molecules are resolved into both parental and recombinant duplexes when treated in vitro with Holliday junction-resolving endonucleases RuvC or T4 endo VII. Taken together with previous observations, these results strongly suggest that joint molecules are double Holliday junctions.

Marker-dependent recombination in T4 bacteriophage. IV. Recombinational effects of antimutator T4 DNA polymerase.

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3'-->5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49)-->3'-->5' exonuclease (gp43)-->DNA polymerase (gp43)-->DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.

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.

Endonuclease VII resolves Y-junctions in branched DNA in vitro.

Endonuclease VII (gp 49 of phage T4) resolves four-way junctions in branched DNAs. We have extended our investigations of the specificity of endo VII and tested its activity with three-way junctions (Y-structures) constructed in vitro. Both 'closed' and 'open' Y-structures were made, absolutely identical in sequence but differing from each other by a single nick in one of the three arms. Pure Y-structures were obtained on a preparative scale by annealing plus and minus strands from two M13mp strains. One strain has an inverted repeat of 2 X 31 nucleotides cloned into the single EcoRI site while in the other strain this repeat is absent. The structures were used in reactions with endo VII, which recognizes the branch point of both structures and introduces a characteristic number of nicks, 3' to the junction in each arm of the structure. Strong and weak sites could be distinguished and the cleavage pattern differed significantly between the two structures. The observed resolution of Y-junctions by endo VII in vitro is compatible with a model for the resolution of recombinant Y-branches in DNA.

Enzymes and Sites of Genetic Recombination: Studies with Gene-3 Endonuclease of Phage T7 and with Site-affinity Mutants of Phage

Branched DNA is an intermediate in genetic recombination (Broker and Lehman 1971; Kemper and Brown 1976; Tsujimoto and Ogawa 1977; Bell and Byers 1979; Ikeda and Kobayashi 1979). One form of branched DNA, the Holliday structure, arises by a reciprocal exchange of strands between duplexes (Holliday 1964). It has recently been shown that gene 49 of bacteriophage T4 specifies or controls an endonuclease (Endo VII) that cleaves Holliday structures (Mizuuchi et al. 1982; Fig. 1). Endo VII is essential for T4 growth; when it is defective, large branched DNA structures accumulate, and these cannot be packaged into mature viral particles (Kemper and Brown 1976; Kemper and Janz 1976). Because bacteriophages T4 and T7 have similar modes of replication, involving complex concatemeric intermediates, high recombination frequencies, and maturation of the DNA during packaging, it seems likely that a similar endonuclease...