Nonpolar nucleobase analogs illuminate requirements for site-specific DNA cleavage by vaccinia topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the +2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the +1T base was not elided by F substitution. We impute a role for +1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the +4G and +3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for +4G and +3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the +1A base of the nonscissile strand; the reaction equilibrium is restored by +1 indole, signifying that base stacking flanking the nick is critical for the religation step. Our findings highlight base isosteres as valuable tools for the analysis of proteins that act on DNA in a site-specific manner.     

Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.     

Site-specific DNA cleavage by vaccinia virus DNA topoisomerase I. Role of nucleotide sequence and DNA secondary structure.

Cleavage of linear duplex DNA by purified vaccinia virus DNA topoisomerase I occurs at a conserved sequence element (5'-C/T)CCTT decreases) in the incised DNA strand. Oligonucleotides spanning the high affinity cleavage site CCCTT at nucleotide 2457 in pUC19 DNA are cleaved efficiently in vitro, but only when hybridized to a complementary DNA molecule. As few as 6 nucleotides proximal to the cleavage site and 6 nucleotides downstream of the site are sufficient to support exclusive cleavage at the high affinity site (position +1). Single nucleotide substitutions within the consensus pentamer have deleterious effects on the equilibria of the topoisomerase binding and DNA cleavage reactions. The effects of base mismatch within the pentamer are more dramatic than are the effects of mutations that preserve base complementarity. Competition experiments indicate that topoisomerase binds preferentially to DNA sites containing the wild-type pentamer element. Single-stranded DNA containing the sequence CCCTT in the cleaved stand is a more effective competitor than is single-stranded DNA containing the complementary sequence in the noncleaved strand.     

Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro.

Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.     

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.     

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.     

Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.     

Receptor proximity, not intermolecular orientation, is critical for triggering T-cell activation

Engagement of antigen receptors on the surface of T-cells with peptides bound to major histocompatibility complex (MHC) proteins triggers T-cell activation in a mechanism involving receptor oligomerization. Receptor dimerization by soluble MHC oligomers is sufficient to induce several characteristic activation processes in T-cells including internalization of engaged receptors and up-regulation of cell surface proteins. In this work, the influence of intermolecular orientation within the activating receptor dimer was studied. Dimers of class II MHC proteins coupled in a variety of orientations and topologies each were able to activate CD4+ T-cells, indicating that triggering was not dependent on a particular receptor orientation. In contrast to the minimal influence of receptor orientation, T-cell triggering was affected by the inter-molecular distance between MHC molecules, and MHC dimers coupled through shorter cross-linkers were consistently more potent than those coupled through longer cross-linkers. These results are consistent with a mechanism in which intermolecular receptor proximity, but not intermolecular orientation, is the key determinant for antigen-induced CD4+ T-cell activation.     

Resolution of a Holliday junction by vaccinia topoisomerase requires a spacer DNA segment 3' of the CCCTT/ cleavage sites

Vaccinia virus DNA topoisomerase catalyzes reso­lution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5′-CCCTT↓, that are opposed within a partially mobile four-way junction. Efficient resolution occurs on a junction with a 10 bp segment of branch mobility (5′-GCCCTTATCG) that extends 4 bp 3′ of the scissile phosphate. Here we report that resolution is decreased when branch mobility is limited to an 8 bp segment extending 2 bp 3′ of the cleavage site and then eliminated when branch mobility is confined to the 6 bp GCCCTT sequence 5′ of the scissile phosphate. We surmise that a spacer region 3′ of CCCTT is needed for simultaneous cleavage at two opposing sites at the junction. Branch mobility is not required for reaction chem­i­stry at a junction, because topoisomerase cleaves a single CCCTT site in a non-mobile four-way junction where the scissile phosphate is at the crossover point. The junction resolvase activity of topo­isomerase may be involved in forming the hairpin telomeres of the vaccinia genome.     

Mechanism of ATP inhibition of mammalian type I DNA topoisomerase: DNA binding, cleavage, and rejoining are insensitive to ATP

A general, unrefined mechanism of type I DNA topoisomerase action involves several steps including DNA binding, single-strand scission, strand passage resealing, and, possibly, readoption of an active enzyme conformation. None of these steps requires an energy cofactor; however, we have shown previously that several mammalian type I topoisomerases are, in fact, inhibited by ATP. In this study, we wanted to examine which steps in the gross topoisomerase mechanism were sensitive or insensitive to ATP. Nitrocellulose filter binding experiments showed that ATP did not interfere with the binding of DNA by the enzyme and that ATP binding by topoisomerase was 5-fold greater in the presence of DNA than in its absence. Agarose gel electrophoresis in the presence or absence of ethidium bromide indicated that resealing was unaffected by added ATP. The addition of the adenine nucleotide did not alter the pattern of camptothecin-stimulated cleavage of DNA, indicating that strand scission was not the point of inhibition. To test whether strand passage or the readoption of an active conformation was an inhibited step, we used a unique DNA topoisomer as substrate. The results argued against readoption of an active enzyme conformation as an ATP-sensitive process.     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

Identification of specific nonbridging phosphate oxygens important for DNA cleavage by human topoisomerase I

Methylphosphonate-bearing oligonucleotides are characterized by the replacement of one of the nonbridging oxygen atoms with a methyl group. While neutralizing the negative charge associated with the phosphodiester at the point of substitution, the methyl group also imparts chirality to the phosphorus atom. Herein we report the synthesis of a number of oligonucleotides containing isomerically pure S(p) and R(p) methylphosphonates at single positions for the purpose of investigating the hydrogen-bonding contacts necessary for human topoisomerase I function. It was possible to correlate these data to the recent X-ray crystal structure of a truncated form of the enzyme and demonstrate a severe decrease of cleavage efficiency when any of the nonbridging oxygen atoms upstream from the cleavage site was removed. Also observed was increased cleavage for oligonucleotides substituted with methylphosphonates downstream from the cleavage site. These effects were shown to be due primarily to alteration of the binding of the modified DNA substrates by human DNA topoisomerase I.     

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway.     

Common sites for recombination and cleavage mediated by bacteriophage T4 DNA topoisomerase in vitro

We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends.     

Sequence requirements for mammalian topoisomerase II mediated DNA cleavage stimulated by an ellipticine derivative.

Various antitumor drugs stabilize DNA topoisomerase II-DNA transient covalent complexes. The complexes distribution along pBR322 DNA was shown previously to depend upon the nature of the drug (Tewey et al. (1984) Science 226, 466-468). The position in pBR322 of DNA cleavage by calf DNA topoisomerase II for 115 such sites stabilized by an ellipticine derivative and the relative frequency of cleavage at most of these sites were determined. The nucleotide sequence surrounding the 25 strongest sites was analyzed and the following ellipticine specific consensus sequence was deduced: 5'-ANCNT(A/G)T.NN(G/C)N(A/G)-3' where cleavage occurs at the indicated mark. A thymine is always present at the 3' end of at least one strand of the strong cleavage sites, and the dinucleotide AT or GT at the 3' end of the break plays a major role in the complex stabilisation. The predictive value of cleavage of the consensus was tested for two regions of SV40 DNA and cleavage was indeed detected at the majority of the sites matching the consensus. Some complexes stabilized by ellipticine are resistant to salt dissociation and this property seems to be correlated with the presence of symmetrical sequences in the cleavage site with a center of symmetry staggered relatively to the center of symmetry of cleavage.     

Local sequence requirements for DNA cleavage by mammalian topoisomerase II in the presence of doxorubicin

Doxorubicin, a DNA-intercalator, is one of several anti-cancer drugs that have been found to stabilizes topoisomerase II cleavage complexes at drug-specific DNA sites. The distribution and DNA sequence environments of doxorubicin-stabilized sites were determined in the SV40 genome. The sites were found to be most concentrated in the major nuclear matrix-associated region and nearly absent in the vicinity of the replication origin including the enhancer sequences in the 21-bp and 72-bp tandem repeats. Among 97 doxorubicin-stabilized sites that were localized at the DNA sequence level, none coincided with any of the 90 topoisomerase II cleavage sites detected in the same regions in the absence of drug. Cleavage at the 90 enzyme-only sites was inhibited by doxorubicin and never stimulated even at low drug concentrations. All of the doxorubicin-stabilized sites had an A at the 3' terminus of at least one member of each pair of strand breaks that would constitute a topoisomerase II double-strand scission. Conversely, none of the enzyme-only sites had an A simultaneously at the corresponding positions on opposite strands. The 3'-A requirement for doxorubicin-stabilized cleavage is therefore incompatible with enzyme-only cleavage and explains the mutual exclusivity of the two classes of sites.     

Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices

A total of 16 oligodeoxyribonucleotides of general sequence 5′-TCTTCTZTCTTTCT-3′, where Z denotes an N-acyl-N-(2-hydroxyethyl)glycine residue, were prepared via solid phase synthesis. The ability of these oligonucleotides to form triplexes with the duplex 5′-AGAAGATAGAAAGA-HEG-TCTTTCTATCTTCT-3′, where HEG is a hexaethylene glycol linker, was tested. In these triplexes, an ‘interrupting’ T:A base pair faces the Z residue in the third strand. Among the acyl moieties of Z tested, an anthraquinone carboxylic acid residue linked via a glycinyl group gave the most stable triplex, whose UV melting point was 8.4°C higher than that of the triplex with 5′-TCTTCTGTCTTTCT-3′ as the third strand. The results from exploratory nuclease selection experiments suggest that a combinatorial search for strands capable of recognizing mixed sequences by triple helix formation is feasible.     

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.     

Cytosine arabinoside lesions are position-specific topoisomerase II poisons and stimulate DNA cleavage mediated by the human type II enzymes.

Cytosine arabinoside (araC) is an important drug used for the treatment of human leukemias. In order to exert its cytotoxic effects, araC must be incorporated into chromosomal DNA. Although specific DNA lesions that involve base loss or modification stimulate nucleic acid cleavage mediated by type II topoisomerases, the effects of deoxyribose sugar ring modification on enzyme activity have not been examined. Therefore, the effects of incorporated araC residues on the DNA cleavage/religation equilibrium of human topoisomerase IIalpha and beta were characterized. AraC lesions were position-specific topoisomerase II poisons and stimulated DNA scission mediated by both human type II enzymes. However, the positional specificity of araC residues differed from that previously reported for other cleavage-enhancing DNA lesions. Finally, additive or synergistic increases in DNA cleavage were observed in the presence of araC lesions and etoposide. These findings broaden the range of DNA lesions known to alter topoisomerase II function and raise the possibility that this enzyme may mediate some of the cellular effects of araC.