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PubMed1984

A close association between sites of DNase I hypersensitivity and sites of enhanced cleavage by micrococcal nuclease in the 5'-flanking region of the actively transcribed ovalbumin gene.

The organization of chromatin was analysed in a segment of the chicken ovalbumin gene extending 6.5 kb upstream from the start site of transcription. Nuclei of chicken oviduct cells and of erythrocytes, and preparations of 'naked' DNA were digested with DNase I and with micrococcal nuclease. The locations of specific nuclease cleavage sites were determined by analyzing the fragments obtained with an indirect end-labeling technique. In oviduct nuclei there are four regions of DNase I hypersensitivity centered at approximately 0.15, 0.80, 3.2 and 6.0 kb upstream from the mRNA cap site. DNase I hypersensitive regions are absent from the 5'-flanking regions of erythrocyte nuclei. Micrococcal nuclease cleavage sites were found that are unique to oviduct nuclei and others that are enhanced in oviduct nuclei, relative to erythrocyte nuclei and to naked DNA. The locations of these micrococcal nuclease cleavage sites are closely associated with the DNase I hypersensitive regions. Nuclease hypersensitivity in the 5'-flanking region of oviduct nuclei reflects alterations in chromatin structure that are specifically correlated with gene expression. Our results suggest the presence at hypersensitive regions of specific proteins which alter the chromatin structure, making the DNA more accessible to nuclease attack.

Animals↗
PubMed1984

Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization.

We have isolated mutants defective in DNA topoisomerases and an endonuclease from the fission yeast Schizosaccharomyces pombe by screening individual extracts of mutagenized cells. Two type I topoisomerase mutants (top1) and three endonuclease mutants (end1) were all viable. The double mutant top1 end1 was also viable and, in its extract, Mg2+- and ATP- dependent type II activity could be detected. Three temperature-sensitive (ts-) mutants having heat-sensitive (hs-) type II enzymes were isolated, and the ts- marker cosegregated with the hs- type II activity. All the ts- mutations fell in one gene (top2) tightly linked to leul in chromosome II. The nuclear division of single top2 mutants was blocked at the restrictive temperature, but the formation of a septum was not inhibited so that the nucleus was cut across with the cell plate. In contrast, the double top1 top2 mutants were rapidly arrested at various stages of the cell cycle, showing a strikingly altered nuclear chromatin region. The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.

Ascomycota↗
PubMed1986

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.

Base Sequence↗
PubMed1987

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.

Animals↗
PubMed1987

A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication.

Priming at the mouse mitochondrial origin of heavy-strand DNA replication is effected by transcripts from the light-strand promoter. The transition from RNA synthesis to DNA synthesis occurs at specific locations between 75 and 165 nucleotides downstream from the transcriptional initiation site. We have identified and partially purified an endonucleolytic activity that cleaves RNA accurately near one of these transition sites; this finding implies a role of specific RNA processing in DNA replication. Cleavage products possess 5'-phosphoryl and 3'-hydroxyl termini. Heterologous assays using mouse or human mitochondrial endoribonuclease with substrates containing the sequences of the human or mouse mitochondrial origins of heavy-strand DNA replication suggest that selection of the cleavage site is guided by sequences adjacent to the actual position of cleavage.

Animals↗
PubMed1988

Characterization of a transcription factor involved in mother cell specific transcription of the yeast HO gene.

The yeast HO gene, which encodes an endonuclease involved in initiating mating type interconversion, is expressed in mother cells but not in daughters. It has been demonstrated that the SWI5 gene, which is an activator of HO expression, plays a critical role in this differential mother/daughter expression of HO. In this paper we describe the cloning and sequencing of the SWI5 gene. The predicted amino acid sequence derived from the cloned SWI5 gene shows homology with the repeated DNA-binding domains ('zinc fingers') of Xenopus transcription factor TFIIIA. A region of the HO promoter involved in the SWI5-dependent transcriptional activation of HO was identified by deletion analysis of the HO promoter in the chromosome, and by testing the ability of HO DNA fragments to activate transcription in the context of a heterologous promoter. The SWI5 gene product was overproduced in yeast from the GAL1-10 promoter, since the SWI5 protein is made at very low levels in wild-type strains, and protein extracts were used to demonstrate that the SWI5 protein binds in vitro to a segment of the HO promoter required for transcriptional activation in vivo.

Amino Acid Sequence↗
PubMed1988

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.

Base Sequence↗
PubMed1988

DNA deoxyribophosphodiesterase.

A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity. The enzyme has a mol. wt of approximately 50,000-55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase). The protein presumably is active in DNA excision repair to remove a sugar-phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation.

Centrifugation, Density Gradient↗
PubMed1989

Resolution of model Holliday junctions by yeast endonuclease: effect of DNA structure and sequence.

The resolution of Holliday junctions in DNA involves specific cleavage at or close to the site of the junction. A nuclease from Saccharomyces cerevisiae cleaves model Holliday junctions in vitro by the introduction of nicks in regions of duplex DNA adjacent to the crossover point. In previous studies [Parsons and West (1988) Cell, 52, 621-629] it was shown that cleavage occurred within homologous arm sequences with precise symmetry across the junction. In contrast, junctions with heterologous arm sequences were cleaved asymmetrically. In this work, we have studied the effect of sequence changes and base modification upon the site of cleavage. It is shown that the specificity of cleavage is unchanged providing that perfect homology is maintained between opposing arm sequences. However, in the absence of homology, cleavage depends upon sequence context and is affected by minor changes such as base modification. These data support the proposed mechanism for cleavage of a Holliday junction, which requires homologous alignment of arm sequences in an enzyme--DNA complex as a prerequisite for symmetrical cleavage by the yeast endonuclease.

Base Sequence↗
PubMed1989

Cruciform cutting endonucleases from Saccharomyces cerevisiae and phage T4 show conserved reactions with branched DNAs.

We have purified a cruciform DNA resolving endonuclease (Endo X3) greater than 1000-fold from crude extracts of mitotically growing Saccharomyces cerevisiae. The enzyme shows high specificity for DNAs with secondary structures and introduces characteristic patterns of staggered 'nicks' in the immediate vicinity of the structure. The following substrates were analyzed in detail: (i) naturally occurring four-way X junctions in cruciform DNA of a supercoiled plasmid; (ii) synthetic four-way X junctions with arms of 9 bp; (iii) synthetic three-way Y junctions with arms of 10 bp; and (iv) heteroduplex loops with 19 nucleotides in the loop. Cleavages were always found in the double stranded portion of the DNA, located immediately adjacent to the junction of the respective structure. The Endo X3 induced cleavage patterns are identical or very similar to the cleavage patterns induced in the same substrates by endonuclease VII (Endo VII) from phage T4. Furthermore, the activity of Endo X3 is completely inhibited in the presence of anti-Endo VII antiserum. Endo X3 has an apparent mol. wt of 43,000 daltons, determined by gel filtration and of approximately 18,000 daltons in SDS--polyacrylamide gels. Maximum activity of the enzyme was obtained in the presence of 10 mM MgCl2 at 31 degrees C in Tris-HCl buffer over a broad pH range with a maximum approximately 8.0. About 70% of maximal activity was obtained when Mg2+ was replaced by equimolar amounts of Mn2+ or Ca2+.

Base Sequence↗
PubMed1990

Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus.

Extracts of calf thymus have been fractionated to reveal a nuclease activity that specifically cleaves model Holliday junctions in vitro. The products of cleavage are unbranched linear duplex DNA molecules. Using synthetic four-way junctions, we show that the major sites of cutting are diametrically opposed, at sites one nucleotide from the base of the junction. Other types of four-way junctions, including pseudo-cruciform structures and cruciforms extruded from supercoiled plasmids, are also cleaved by the nuclease. The Mr of the partially purified activity, determined by gel filtration, is approximately 75,000. The calf thymus enzyme provides the first example of an endonuclease from a higher eukaryote that acts specifically on branch points in DNA, and indicates that junction-resolving proteins are normal constituents of somatic cells.

Animals↗
PubMed1991

Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure.

Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease.

An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae. A collection of temperature-sensitive strains was screened using this assay, and a mutant lacking the activity was found. The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere. Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus. The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein. The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases. A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination. A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes. The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.

Amino Acid Sequence↗
PubMed1992

Unusual stability of recombination intermediates made by Escherichia coli RecA protein.

The structure and stability of recombination intermediates made by RecA protein have been investigated following deproteinization. The intermediates consist of two duplex DNA molecules connected by a junction, as visualized by electron microscopy. Although we expected the structures to be highly unstable due to branch migration of the junction, this was not the case. Instead, we found that the intermediates were stable at 37 degrees C. At 56 degrees C, greater than 60% of the intermediates remained after 6 h of incubation. Only at higher temperatures was significant branch migration observed. This unexpected stability suggests that the formation of extensive lengths of heteroduplex DNA in Escherichia coli is likely to require the continued action of proteins, and does not occur via spontaneous branch migration. We show that heteroduplex DNA may be formed in vitro by ATP-dependent strand exchange catalysed by RecA protein or by the RuvA and RuvB proteins of E. coli.

Bacterial Proteins↗
PubMed1992

Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme.

The DNA binding activity of Fos and Jun is regulated in vitro by a post-translational mechanism involving reduction-oxidation. Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun. Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref-1) recently purified from Hela cells, stimulates AP-1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity. Here we demonstrate that the protein product of the ref-1 gene stimulates the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kappa B, Myb and members of the ATF/CREB family. Furthermore, immunodepletion analysis indicates that Ref-1 is the major AP-1 redox activity in Hela nuclear extracts. Interestingly, Ref-1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity. However, the redox and DNA repair activities of Ref-1 can, in part, be distinguished biochemically. This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes.

Amino Acid Sequence↗
PubMed1993

Dissociation of synthetic Holliday junctions by E. coli RecG protein.

The RecG protein of Escherichia coli is needed for normal levels of recombination and for repair of DNA damaged by ultraviolet light, mitomycin C and ionizing radiation. The true extent of its involvement in these processes is masked to a large degree by what appears to be a functional overlap with the products of the three ruv genes. RuvA and RuvB act together to promote branch migration of Holliday junctions, while RuvC catalyses the resolution of these recombination intermediates into viable products by endonuclease cleavage. In this paper, we describe the overproduction and purification of RecG and demonstrate that the overlap extends to the biochemistry. We show that the 76 kDa RecG protein is a DNA-dependent ATPase, like RuvB. Using gel retardation assays we demonstrate that it binds specifically to a synthetic Holliday junction, like RuvA and RuvC. Finally, we show that in the presence of ATP and Mg2+, RecG dissociates these junctions to duplex products, like RuvAB. We suggest that RecG and RuvAB provide alternative activities than can promote branch migration of Holliday junctions in recombination and DNA repair.

Adenosine Triphosphatases↗
PubMed1993

The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.

I-TevI, a double-strand DNA endonuclease encoded by the mobile td intron of phage T4, has specificity for the intronless td allele. Genetic and physical studies indicate that the enzyme makes extensive contacts with its DNA substrate over at least three helical turns and around the circumference of the helix. Remarkably, no single nucleotide within a 48 bp region encompassing this interaction domain is essential for cleavage. Although two subdomains (DI and DII) contain preferred sequences, a third domain (DIII), a primary region of contact with the enzyme, displays much lower sequence preference. While DII and DIII suffice for recognition and binding of I-TevI, all three domains are important for formation of a cleavage-competent complex. Mutational, footprinting and interference studies indicate predominant interactions of I-TevI across the minor groove and phosphate backbone of the DNA. Contacts appear not to be at the single nucleotide level; rather, redundant interactions and/or structural recognition are implied. These unusual properties provide a basis for understanding how I-TevI recognizes T-even phage DNA, which is heavily modified in the major groove. These recognition characteristics may increase the range of natural substrates available to the endonuclease, thereby extending the invasive potential of the mobile intron.

Base Sequence↗