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.     

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.     

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.     

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 IV enhances base excision repair of endonuclease III from Methanobacterium thermoautotrophicum

Damaged DNA strands are repaired by base excision (BER) in organisms, a process initiated by repair enzymes, which include DNA glycosylases and endonucleases. We expressed and characterized two putative endonuclease genes from Methanobacterium thermoautotrophicum, Mt0764 and Mt1010, encoding homologues of endonuclease III (endo III) and endonuclease IV (endo IV) of Escherichia coli. The Mt0764 and Mt1010 proteins showed endo III activity by removing thymine glycol from DNA strand and AP endonuclease activity, respectively. The Mt0764 protein not only cleaved the oligonucleotide duplex, containing a thymine glycol/adenine pair efficiently, but also showed activity on the 8-oxoguanine-containing oligonucleotide duplex. In this study, we report upon the stimulation of endo III activity by endo IV using two recombinant proteins (Mt1010 and Mt0764) from M. thermoautotrophicum. Mt1010 stimulated the DNA glycosylase activity of Mt0764 for DNA substrates containing 8-oxoguanine residues and increasing the formation of the Mt0764 protein-DNA complex. The interaction between Mt1010 and Mt0764 was observed by using an in vitro binding assay. These results suggest that association between endo III and endo IV may occur in vivo, and this contributes to efficient base excision repair for the oxidative damage of DNA.     

The assay and isolation of DNA rings using an ATP-dependent endonuclease.

The ATP-dependent endonuclease from Hemophilus influenzae is relatively inactive on closed or open DNA rings, yet rapidly hydrolyzes single- or double-chained linear DNA. This enzyme in combination with an exonuclease (exo VII) has been shown to spare various circular DNA molecules including those having single-chain regions of significant length. However, rings containing single-chained regions are broken at a rate depending on the length of these regions. By admixing a linear DNA of alternate radiolabel, a simple assay for DNA rings has been developed. The application of this procedure to the assay of folded rings from Drosophila DNA is demonstrated.     

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.     

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.     

In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate.

The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.     

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 IS10 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 IS10 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 IS10 insertion. The expression of gene 49 in E. coli provides a particularly useful tool for the analysis of mutational events.     

Cleavage map of bacteriophage T4 cytosine-containing DNA by sequence-specific endonucleases SalI and KpnI.

Cytosine-containing T4 DNA from endoII- endoIV- dCTPase- alc2 phage grown in a sup+ rB- mB- host is cleaved by endo R.EcoRI and endo R.HindIII to greater than 40 fragments and by endo R.SalI and endo R.KpnI to 8 and 6 fragments, respectively. The latter two fragment sets have been correlated to each other to produce a cleavage map of the genome. The sum of the molecular weights of the fragments calculated from electrophoretic mobility in agarose gels yields a genome molecular weight for cytosine-containing T4 DNA of 105 x 10(6).     

DNA-binding properties of the major core protein of adenovirus 2.

The major adenovirus core protein (P.VII) binds to various species of duplex and single-stranded DNA molecules as a linear function of P.VII concentration. P.VII progressively condenses 32S Ad2 DNA into rapidly sedimenting forms having an S value of around 2,280. P.VII does not coat DNA like cytochrome C, instead DNA-protein beads are visualized in the electron microscope at low protein concentration. These beads appear to interact forming larger structures and at high P.VII concentrations the DNA molecule becomes highly compacted. Analysis of DNA fragments formed after digestion of P.VII-DNA complexes and isolated cores with micrococcal nuclease suggest that the organization of the DNA in the two structures is essentially identical. The initial P.VII and DNA interaction is sensitive to both ionic and hydrophobic environments, whereas the in vitro DNA-P.VII complexes are extremely stable and are not disrupted in the presence of 3 M NaCl, 1% sarcosyl or 5% deoxycholate. Properties of these in vitro DNA-protein VII complexes share striking similarities to isolated viral core particles.     

Analysis of the alpha-satellite DNA from African green monkey cells by restriction nucleases.

By the use of restriction endonucleases the organization of the alpha-satellite DNA from African green monkey cells (Cercopithecus aethiops) has been analyzed. With endo R-HindIII, endo R-AluI and with endo R-EcoRI at conditions of low salt and high pH (endo R-EcoRI) all of the satellite was digested while only a part of the satellite was cleaved with endo R-Bsu and endo R-EcoRI under standard conditions. With each of the four nucleases a series of fragments was formed which were multiplies in size of a basic repeat unit linked in tandem arrays in the intact satellite. The quantitative evaluation of the digestion with each nuclease as well as with combinations of two nucleases yielded information about the distribution of the cleavage sites. While the arrangement of the endo R-HindIII cleavage sites conforms to a random distribution across the entire satellite, the results from the endo R-Bsu and endo R-EcoRI cleavage patterns are consistent with a picture where the cleavage sites are clustered in fractions of the satellite. Since endo R-AluI recognizes the central four nucleotide pairs of the endo R-HindIII cleavage site, the redigestion of the endo R-HindIII dimer with endo R-AluI gave information about the distribution of mutations in the satellite. The results of these experiments together with the comparison of the sequence divergence determined from digestion with endo R-HindIII and endo R-EcoRI lend support to the hypothesis that mutations have affected all bases in the satellite evenly. The gamma-satellite, another fraction of the African green monkey DNA, could be separated by Ag+/CsSO4 density gradient centrifugation into two components. With the three restriction nucleases used both components gave a background of fragments of heterogenous length on gel electrophoresis with some faint bands of no apparent regularity in one case.     

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.     

Conformational flexibility in DNA structure and its implications in understanding the organization of DNA in chromatin.

X-ray crystallographic studies of drug-nucleic acid crystalline complexes have suggested that DNA first bends or 'kinks' before accepting an intercalative drug or dye. This flexibility in DNA structure is made possible by altering the normal C2' endo deoxyribose sugar puckering in B DNA to a mixed sugar puckering pattern of the type C3' endo (3'-5') C2' endo and partially unstacking base pairs. A kinking scheme such as this would require minimal sterochemical rearrangement and would also involve small energies. This has prompted us to ask more generally if a conformational change such as this could be used by proteins in their interactions with DNA. Here we describe an interesting superhelical DNA structure formed by kinking DNA every ten base pairs. This structure may be used in the organization of DNA within the nucleosome structure in chromatin.     

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII.

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.     

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.     

Enzymatic mutation detection: enrichment of heteroduplexes from hybrid DNA mixtures by cleavage-deficient GST-tagged endonuclease VII.

A method for the enrichment of heteroduplex DNAs from hybrid DNA mixtures by endonuclease VII is reported. The procedure is based on the ability of a GST-fused cleavage-deficient mutant endonuclease VII (EVII-N62D(GST)) to bind to mismatching nucleotides in heteroduplex DNAs identical to the wild-type enzyme. The GST tag was used for stable immobilisation of the protein to Glutathione Sepharose 4B. This enables the material to withstand the repeated rounds of binding steps required for enrichment of heteroduplex molecules from appropriate samples.