DNA topoisomerase II cleavage of telomeres in vitro and in vivo.

In this work, we have analyzed the reactivity of DNA topoisomerase II with telomeric DNA both in vitro and in vivo. Topoisomerase II cleavage reactions were performed on the tandem repeats of telomeric DNA. Analysis of this DNA on sequencing gels revealed that DNA topoisomerase II is catalytically active in cleaving the telomere DNA repeat. The topoisomerase II cleavage site is 5'TTAGG*G3' (cleavage site marked by the asterisk) and since telomere DNA is a tandem array of the above sequence, topoisomerase cleavage sites could exist every six base pairs. Detection of topoisomerase II cleavages was strongly dependent upon one specific topoisomerase II poison, etoposide (VP-16). A number of other topoisomerase II poisons were tested but did not stimulate cleavage activity at the telomere repeat. We have also analyzed the association of endogenous topoisomerase II with chromosomal telomeric DNA in HeLa cells. The in vivo complex of enzyme (ICE) bioassay was used to isolate topoisomerase II-DNA covalent complexes. In consistence with in vitro cleavage data, endogenous topoisomerase II-telomeric DNA complexes were detected in only etoposide-treated HeLa cells.

Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells.

The cytosine analog 5-aza-2'-deoxycytidine is a potent inhibitor of DNA methyltransferase. Its cytotoxicity has been attributed to several possible mechanisms including reexpression of growth suppressor genes and formation of covalent adducts between DNA methyltransferase and 5-aza-2'-deoxycytidine-substituted DNA which may lead to steric inhibition of DNA function. In this study, we use a panel of human breast cancer cell lines as a model system to examine the relative contribution of two mechanisms, gene reactivation and adduct formation. Estrogen receptor-negative cells, which have a hypermethylated estrogen receptor gene promoter, are more sensitive than estrogen receptor-positive cells and underwent apoptosis in response to 5-aza-2'-deoxycytidine. For the first time, we show that reactivation of a gene silenced by methylation, estrogen receptor, plays a major role in this toxicity in one estrogen receptor-negative cell line as treatment of the cells with anti-estrogen-blocked cell death. However, drug sensitivity of other tumor cell lines correlated best with increased levels of DNA methyltransferase activity and formation DNA.DNA methyltransferase adducts as analyzed in situ. Therefore, both reexpression of genes like estrogen receptor and formation of covalent enzyme. DNA adducts can play a role in 5-aza-2'-deoxycytidine toxicity in cancer cells.

Molecular structure of the halogenated anti-cancer drug iododoxorubicin complexed with d(TGTACA) and d(CGATCG).

4'-Deoxy-4'-iododoxorubicin, a halogenated anthracycline derivative, is an anticancer agent currently under Phase II clinical trials. In preclinical studies, it has demonstrated significantly reduced levels of cardiotoxicity compared to currently employed anthracyclines. It also has modified pharmacological properties resulting in an altered spectrum of experimental antitumor activity. The iodine atom at the 4' position of the sugar ring reduces the basicity and enhances the lipophilicity of this compound as compared to related anthracycline drugs. We report here single crystal X-ray diffraction studies of the complexes of 4'-deoxy-4'-iododoxorubicin with the hexanucleotide duplex sequences d(TGTACA) and d(CGATCG) at 1.6 and 1.5 A, respectively. The iodine substituent does not alter the geometry of intercalation as compared to previously solved anthracycline complexes, but appears to markedly affect the solvent environment of the structures. This could have consequences for the interaction of this drug with DNA and DNA binding proteins in cells.

Analysis of eukaryotic topoisomerase II cleavage sites in the presence of the quinolone CP-115,953 reveals drug-dependent and -independent recognition elements.

The quinolone derivative CP-115,953 [6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] has been shown to induce eukaryotic topoisomerase II-mediated breaks in DNA, producing cleavage patterns that are distinct from those induced by the anticancer drugs amsacrine, etoposide, and teniposide. High levels of the quinolone have been found to inhibit topoisomerase II activity via an interaction with the enzyme and not by DNA unwinding. Topoisomerase II cleavage sites were analyzed on nine DNA fragments, and 85 quinolone-induced sites were sequenced, as well as 86 amsacrine and 134 teniposide sites. A consensus sequence was derived for the quinolone sites that is different from those reported for other drugs; however, because topoisomerase II cleavage sites are double-stranded but not palindromic, different consensus sequences are not easily compared. For this reason, a new, double-stranded, consensus sequence method, the "unique-base analysis," was developed; this was applied to the quinolone sites as well as six other large sets of topoisomerase II sites determined in the absence or presence of drugs. For each of the seven sets of sites, conserved bases were found in the 16-base region spanning positions -6 to +10, relative to the enzyme cleavage site (DNA breakage between -1 and +1). The conserved bases were virtually identical in the regions flanking the cleavage site for all seven data sets. In contrast, the base preferences identified proximal to the cleavage sites were unique to the drug tested. These observations suggest that the selection of cleavage sites by topoisomerase II involves both enzyme-dependent and drug-dependent recognition elements. The single most preferred base in the quinolone sites was a cytosine at -1; the same preference was found with teniposide, and 60 of the 85 quinolone sites co-localized with teniposide sites.

Determination of 5' and 3' DNA triplex interference boundaries reveals the core DNA binding sequence for topoisomerase II.

Previous studies have shown that formation of intermolecular DNA triplexes at sequences that overlap protein binding sites inhibits DNA binding by these proteins. We show that DNA cleavage by eukaryotic topoisomerase II is blocked by triplex formation at sites overlapping and adjacent to the triple binding site. To map precisely the boundaries of triplex interference, we constructed a vector containing enzyme binding sites of different lengths and flanked both 5' and 3' by DNA triplexes. We call this method Triplex Interference Mapping by Binding Element Replacement (TIMBER). Triplex regions within 3 bases 5' or 7 bases 3' of cleavage sites blocked DNA cleavage; triplex formation outside of this region had no effect upon cleavage activity. We conclude that topoisomerase II binding requires unhindered access to the major groove of a duplex DNA binding site in this 10-base region. In addition, the inclusion of topoisomerase II inhibitors yielded the same results for the triplex interference assays despite alterations in DNA cleavage site selection. The statistical analyses of over 500 topoisomerase II cleavage sites (in the presence or absence of inhibitors) suggest a model consistent with the region spanning -3 to +7 (relative to the cleavage site) containing most of the base-specific contacts for topoisomerase II. This triplex interference assay may prove valuable in the characterization of DNA binding sites for other proteins as well, particularly in conjunction with deletion analysis.

Z-DNA binding protein from chicken blood nuclei.

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

Eukaryotic topoisomerase II cleavage of parallel stranded DNA tetraplexes.

A guanine-rich single-stranded DNA from the human immunoglobulin switch region was shown by Sen and Gilbert [Nature, (1988) 334, 364-366] to be able to self-associate to form a stable four-stranded parallel DNA structure. Topoisomerase II did not cleave the single-stranded DNA molecule. Surprisingly, the enzyme did cleave the same DNA sequence when it was annealed into the four-stranded structure. The two cleavage sites observed were the same as those found when this DNA molecule was paired with a complementary molecule to create a normal B-DNA duplex. These cleavages were shown to be protein-linked and reversible by the addition of salt, suggesting a normal topoisomerase II reaction mechanism. In addition, an eight-stranded DNA molecule created by the association of a complementary oligonucleotide with the four-stranded structure was also cleaved by topoisomerase II despite being resistant to restriction endonuclease digestion. These results suggest that a single strand of DNA may possess the sequence information to direct topoisomerase II to a binding site, but the site must be base paired in a proper manner to do so. This demonstration of the ability of a four-stranded DNA molecule to be a substrate for an enzyme further suggests that these DNA structures may be present in cells.

A predictive model for DNA recognition by the herpes simplex virus protein ICP4.

The herpes simplex virus (HSV) type 1 immediate early protein ICP4 is an essential regulatory enzyme that binds DNA directly in order to stimulate or repress gene expression. The degree of transaction is related to the locations and affinities of the ICP4 binding sites. A number of binding sites have been identified; some sites showed obvious homology to one another, and these were called consensus ICP4 binding sites. Other binding sites did not appear to be related, and these were termed non-consensus sites. We hypothesized, however, that a single model could describe all ICP4 binding sites, given the appropriate characterizations of sites. We performed statistical analyses on a set of ICP4 binding sites and found that the bases important for defining binding were located within a 13 base region. Missing contact analyses on several high-affinity binding sites revealed the same 13 base region as important for critical protein-DNA contacts. From these data we derived the consensus sequence RTCGTCNNYNYSG, where R is purine, Y is pyrimidine, S is C or G, and N is any base. In addition, we found that a better profile for ICP4 binding sites involves use of a matrix of base proportions from the binding site data; sites are analyzed by calculating the Matrix Mean score. We show that this Matrix Mean model could accurately predict the locations of novel ICP4 binding sites. Finally, we analyzed the entire HSV-1 genome for potential ICP4 binding sites and speculate about what these results suggest for the role of ICP4 in viral gene regulation.

Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats.

Alternating purine-pyrimidine sequences (RY repeats) demonstrate considerable homology to the consensus sequence for vertebrate topoisomerase II (Spitzner and Muller (1988) Nucleic Acids Res. 16: 1533-1556). This is shown below and positions that can match are underscored. RYRYRYRYRYRYRYRYRY = alternating purine-pyrimidine 18 bp RNYNNCNNGYNGKTNYNY = topoisomerase II consensus sequence (R is purine, Y is pyrimidine, K is G or T.) Topoisomerase II cleavage reactions were performed (in the absence of inhibitors) on a plasmid containing a 54 base RY repeat and the single strong cleavage site mapped to the RY repeat. Analysis of this DNA on sequencing gels showed that the enzyme cleaved a number of sites, all within the 54 base pair RY repeat. Topoisomerase II also made clustered cleavages within other RY repeats that were examined. Quantitative analysis of homology to the consensus sequence, as measured by the match of a site to a matrix of base proportions from the consensus data base (the matrix mean), showed that both the locations and the frequencies of cleavage sites within RY repeats were proportional to homology scores. However, topoisomerase II cleaved RY repeats preferentially in comparison to non-RY sites with similar homology scores. The activity of the enzyme at RY repeats appears to be proportional to the length of the repeat; additionally, GT, AC and AT repeats were better substrates for cleavage than GC repeats.

Application of a degenerate consensus sequence to quantify recognition sites by vertebrate DNA topoisomerase II.

A consensus sequence has been derived for vertebrate topoisomerase II cleavage of DNA (Spitzner, J. R. and Muller, M. T. (1988) Nucleic Acid. Res. 16, 5533-5556). An independent sample of 65 topoisomerase II sites (obtained in the absence of topoisomerase II inhibitors) was analyzed and found to match the consensus sequence as well as enzyme sites determined in the presence of the anti-tumor drug 4'-(9-acridinyl-amino)-methanesulfon-m-anisidide (m-AMSA). As originally described, conventional application of the consensus sequence afforded accuracy in the prediction of the locations but not the frequencies of topoisomerase II cleavages. In the present report, we describe a new method which quantitatively discriminates sites from nonsites, called the 'matrix mean' method (the mean match of a site to the matrix of base proportions from the original consensus sequence derivation). Furthermore, we derived a second method, called the 'unique score' model, which predicts frequency of topoisomerase II activity at a cleavage site. In the unique score method both DNA strands of a site are examined to determine the total number of the consensus positions that match on at least one strand of a potential site. From the new data base of 65 topoisomerase II sites, cleavages were scored for relative cleavage strength. Linear regression analysis showed a significant (p less than 0.01) correlation between the unique score and cleavage strength. The study was extended to show that the unique score model accurately and quantitatively predicts topoisomerase II sites either in the absence or presence of m-AMSA using the same consensus sequence.

Single-strand DNA cleavages by eukaryotic topoisomerase II.

A new purification method for eukaryotic type II DNA topoisomerase (EC 5.99.1.3) is described, and the avian enzyme has been purified and characterized. An analysis of the cleavage reaction has revealed that topoisomerase II can be trapped as a DNA-enzyme covalent complex containing DNA with double-stranded and single-stranded breaks. The data indicate that DNA cleavage by topoisomerase II proceeds by two asymmetric single-stranded cleavage and resealing steps on opposite strands (separated by 4 bp) with independent probabilities of being trapped upon addition of a protein denaturant. Single-strand cleavages were directly demonstrated at both strong and weak topoisomerase II sites. Thus, a match to the vertebrate topoisomerase II consensus sequence (sequence; see text) (N is any base, and cleavage occurs between -1 and +1) [Spitzner, J.R., & Muller, M.T. (1988) Nucleic Acids Res. 16, 5533-5556)] does not predict whether a cleavage site will be single stranded or double stranded; however, sites cleaved by topoisomerase II that contain two conserved consensus bases (G residue at +2 and T at +4) generally yield double-strand cleavage whereas recognition sites lacking these two consensus elements yield single-strand cleavages. Finally, single-strand cleavages with topoisomerase II do not appear to be an artifact caused by damaged enzyme molecules since topoisomerase II in freshly prepared, crude extracts also shows the property of single-strand cleavages.

A consensus sequence for cleavage by vertebrate DNA topoisomerase II.

Topoisomerase II, purified from chicken erythrocytes, was reacted with a large number of different DNA fragments and cleavages were catalogued in the presence and absence of drugs that stabilize the cleavage intermediate. Cleavages were sequenced to derive a consensus for topoisomerase II that predicts catalytic sites. The consensus is: (sequence; see text) where N is any base and cleavage occurs at the indicated mark between -1 and +1. The consensus accurately predicts topoisomerase II sites in vitro. This consensus is not closely related to the Drosophila consensus sequence, but the two enzymes show some similarities in site recognition. Topoisomerase II purified from human placenta cleaves DNA sites that are essentially identical to the chicken enzyme, suggesting that vertebrate type II enzymes share a common catalytic sequence. Both viral and tissue specific enhancers contain sites sharing strong homology to the consensus and endogenous topoisomerase II recognizes some of these sites in vivo.