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Query: UNIPROT:P06889 (Mol)
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Endonuclease VII of phage T4 resolves Holliday structures in vitro by nicking pairs of strands across the junction. We report here analyses of this reaction between endonuclease VII and a Holliday structure analogue, made in vitro from synthetic oligonucleotides. The enzyme cleaves the structure in a non-concerted way and nicks each strand independently. Combinations of nicks with counter-nicks in strands across the junction resolve the construct. The specificity of the enzyme for DNA secondary structures was tested with a series of branched molecules made from oligonucleotides with the same nucleotide sequence in one strand. Results show that the number, location and relative cleavage efficiencies depend largely on the local nucleotide sequence, rather than on the branch type. In particular, endonuclease VII cleaves a complete four-armed cruciform as efficiently as a three-armed Y-junction or its derivatives, a semi-Y, a fork with two single-strand overhangs, a single-strand overhang, and a nicked DNA. However, exchange or addition of one or more nucleotides within the cleavage area flanking the structural signal for endonuclease VII strongly affects the cleavage pattern as well as their relative efficiency of usage. Examples with a single-stranded overhang are presented and show in summary that the enzyme has a fivefold preference for pyrimidines rather than purines.
J Mol Biol 1992 Feb 05
PMID:T4 endonuclease VII resolves cruciform DNA with nick and counter-nick and its activity is directed by local nucleotide sequence. 154 8

Four-way DNA junctions are thought to be important intermediates in a number of recombination processes. Resolution of these junctions occurs by cleavage of two strands of DNA to generate two duplex molecules. The interaction between DNA junctions and resolving enzymes appears to be largely structure-specific, reflecting a molecular recognition on a significant scale. We propose a working model for this interaction that takes account of the present state of knowledge of the structure of the DNA junction, and the substrate requirements of the enzymes. We note that three different enzymes introduce cleavages at phosphodiester bonds that are presented on one side of the molecule, suggesting that the enzymes selectively interact with this face of the junction. By forcing a junction of constant sequence to adopt one or other of the two possible antiparallel isomers, we show that the junction is cleaved in such a way as to suggest a constant mode of interaction with the protein that is dependent on structure rather than sequence. We propose that the feature that is recognized is a mutual inclination of two DNA helices at approximately 120 degrees. We show that a number of DNA substrates that contain similar inclined helices, such as a three-way junction, bulged duplexes and a duplex that is curved because of repeated runs of oligoadenine sequences, are each cleaved by phage T4 endonuclease VII. This mode of DNA-protein interaction could be significant in either recombination or DNA repair processes.
J Mol Biol 1991 Oct 20
PMID:Model for the interaction of DNA junctions and resolving enzymes. 165 36

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.
J Mol Biol 1990 Apr 20
PMID:Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions. 215 65

Two monoclonal antibodies (2D3 and 4B4) have been raised against a stable cruciform DNA structure containing the 27-base pair palindrome of the SV40 origin of replication on one strand and an unrelated 26-base pair palindrome on the complementary strand (pRGM 21 x pRGM 29) and have been shown to recognize conformational determinants specific to cruciform DNA structures (Frappier, L., Price, G.B., Martin, R. G., and Zannis-Hadjopoulos, M. (1987) J. Mol. Biol. 193, 751-758). To define the region(s) of the cruciform that is recognized by these antibodies, we examined the ability of 2D3 and 4B4 to protect the single-stranded tips of the loops or the four-way junctions at the base of the stem of stable cruciform molecules against cleavage by mung bean nuclease or T7 endonuclease 3, respectively. Both antibodies were found to protect two of the four elbow-like structures at the base of the cruciform from T7 endonuclease 3 cleavage, but not the tips of the cruciform arms from mung bean nuclease cleavage. Also, predigestion of the cruciform with mung bean nuclease did not affect the binding of either antibody. In addition, 2D3 bound to a cruciform and a T-shaped structure involving the palindromic sequence at the cloning site of pUC7, which is completely unrelated in sequence to the palindrome of pRGM 21 x pRGM 29, and protected the base of these stem-loop structures against cleavage by T4 endonuclease VII. These results indicate that 2D3 and 4B4 bind at or near the base of the cruciform molecules and that, at least for 2D3, binding is independent of DNA sequence.
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PMID:Characterization of the binding specificity of two anticruciform DNA monoclonal antibodies. 246 59

This paper describes the nucleotide sequences of three spontaneous mutations in a suppressor gene of phage T4 tRNA(Ser). They are duplications of the anticodon and variable arms of the tRNA(Ser) molecule. One is a 34-nucleotide direct repeat of the wild-type sequence. The remaining two have reciprocal structures, with each containing 35-nucleotide inverted and direct repeats of the wild-type sequence. One of the latter mutations is frequent and was present in multiple isolates. All three duplications are unstable, and several revertants of each were sequenced. Most of the revertants had the wild-type nucleotide sequence; however, one had imprecisely removed the duplicated residues, leaving four new nucleotides compared to the wild-type sequence. These mutations represent significant genetic events with regard to their high rates and their gross structural alterations. As to their origin, the mutations can be described as the end-products of endonuclease cleavage of DNA at regions of potential secondary structure and subsequent DNA synthesis. The secondary structure contains four base-paired stems that emerge from duplex DNA. These stems encode the anticodon and variable arm regions of the tRNA(Ser) molecule. The cleavage sites mimic the known substrate of T4 endonuclease VII, an enzyme previously noted for its ability to resolve Holliday-like DNA intermediates.
J Mol Biol 1988 Nov 05
PMID:Specific duplications fostered by a DNA structure containing adjacent inverted repeat sequences. 285 Oct 51

The sensitivity of the ColE1 cruciform to four enzyme and chemical probes of secondary structure has been studied as a function of plasmid topology. Purified topoisomers of pColIR515 have been probed with S1 nuclease, Bal31 nuclease, phage T4 endonuclease VII or osmium tetroxide, and site-specific reaction quantified. Closely similar profiles of reactivity as a function of linking difference were obtained for each probe. Electrophoresis of the pure topoisomers on polyacrylamide/agarose gels revealed a discontinuity in migration as a function of linking difference. Above a threshold linking difference, topoisomers exhibit pronounced reduction in mobility. The linking difference at which this band shift is found correlates precisely with that required for site-specific reaction with the four probes. We conclude that both probing and topological methods are valuable in the study of cruciform structure in supercoiled DNA. The band shift has been measured with accuracy to allow the calculation of the twist change that accompanies the transition, corresponding to delta Tw = -3.2 +/- 0.1. Using this value together with the critical linking difference we calculate a free energy of formation for this structure delta G = 18.4 +/- 0.5 kcal mol-1 (1 kcal = 4.184 kJ).
J Mol Biol 1984 Nov 25
PMID:Thermodynamics of the ColE1 cruciform. Comparisons between probing and topological experiments using single topoisomers. 609 58

Endonuclease VII, the product of phage T4 gene 49, has been shown previously to resolve Holliday structures in vitro. Two different processes, genetic recombination and multiplicity reactivation are presumed to have Holliday structure intermediates. Other workers have shown that genetic recombination is reduced in a gene 49 mutant infection. However, in the present study, multiplicity reactivation of UV-irradiated ts or amber mutant phage defective in gene 49 was nearly identical to that of UV-irradiated wild-type phage T4. Thus endonuclease VII is not thought to be essential for multiplicity reactivation of phage T4.
Mol Gen Genet 1983
PMID:Gene 49 endonuclease VII is not essential for multiplicity reactivation of bacteriophage T4. 631 42

Bacteriophage T4 endonuclease VII is one of a class of structure-selective enzymes that resolve helical branchpoints in DNA molecules. The sequence of this protein suggests a modular organisation. We have expressed a synthetic gene encoding endonuclease VII, which has been used in a directed mutagenesis exercise, with the aim of understanding the role of different sections of the protein sequence. Towards the N-terminal end of the protein lies a section of polypeptide in which four cysteine residues distributed in a CxxC--CxxC pattern co-ordinate one atom of zinc. The N-terminal section composed of amino acid residues 1 to 65 isolated from the remaining C-terminal section also binds one mole of zinc, suggesting that this region folds autonomously. Mutation shows that the outer cysteine residues are essential for zinc binding, while the inner cysteine residues are partially degenerate in that either one of the two (but not both) can be replaced while retaining some zinc. The activity as a junction-resolving enzyme correlated qualitatively with the presence of the zinc. In the C-terminal part of the protein lies a section that is 48% identical with a sequence found in the DNA repair protein T4 endonuclease V. We can replace the section of T4 endonuclease VII with the corresponding sequence from T4 endonuclease V with no change in the pattern of cleavage on four-way junctions. The evidence supports a modular construction for T4 endonuclease VII.
J Mol Biol 1995 Oct 06
PMID:The modular character of a DNA junction-resolving enzyme: a zinc-binding motif in bacteriophage T4 endonuclease VII. 756 77

Bacteriophage T7 endonuclease I is a resolving enzyme that selectively cleaves four-way DNA junctions, and related branched species. We have isolated mutants of this protein that retain full structural selectivity of binding to four-way junctions, but which are completely inactive as nucleases. This is consistent with a divisibility of structure-selective binding and catalysis. The mutations that inactivate endonuclease I as a nuclease are clustered into the second quarter of the primary sequence, a region that displays some sequence similarity with the related junction-resolving enzyme endonuclease VII from bacteriophage T4. This suggests that these residues may form the active site of these enzymes. The configuration of the helical arms of the junction bound by mutant endonuclease I has been investigated by gel electrophoretic methods. We find that the junction is bound in the presence or absence of magnesium ions, and that the global structure of the bound form is apparently identical with or without cations. The patterns of mobilities suggest that the structure of the junction becomes perturbed by the binding of the protein.
J Mol Biol 1995 Feb 10
PMID:Binding of the junction-resolving enzyme bacteriophage T7 endonuclease I to DNA: separation of binding and catalysis by mutation. 785 9

T4 endonuclease VII is an enzyme that cleaves four-way junctions and other branched DNA structures. We now show that a DNA duplex containing a single cis-diammineplatinum (II) adduct at either GpG or ApG sequences is a good substrate for precise cleavage by this enzyme. The GpG platinated DNA was cleaved more efficiently than the equivalent ApG platinated DNA. A duplex containing a trans-diammineplatinum (II) adduct at a GpTpG sequence was refractory to cleavage. We suggest that local kinking of the helix due to the chemical modification by cis-dichlorodiammineplatinum (II) could be responsible for the sensitivity of the DNA to attack by endonuclease VII, in common with the structures of many other substrates for this enzyme. These results are in keeping with a general role for T4 endonuclease VII in the repair of DNA structure, and a mechanism of substrate recognition that is based on sensitivity to axial inclination.
J Mol Biol 1993 Sep 05
PMID:T4 endonuclease VII cleaves DNA containing a cisplatin adduct. 837 95


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