Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.     

Induction of cleavage in topoisomerase I c-DNA by topoisomerase I enzymes from calf thymus and wheat germ in the presence and absence of camptothecin.

In this study, we further examined the sequence selectivity of camptothecin in mammalian topoisomerase I cDNA from human and Chinese hamster. In the absence of camptothecin, almost all the bases at the 3'-terminus of cleavage sites are T for calf thymus and wheat germ topoisomerase I. In addition, wheat germ topoisomerase I exhibits preference for C (or not T) at -3 and for T at -2 position. As for camptothecin-stimulated cleavage with topoisomerase I, G (or not T) at +1 is an additional strong preference. This sequence selectivity of camptothecin is similar to that previously found in SV40 DNA, suggesting that camptothecin preferentially interacts with topoisomerase I-mediated cleavage sites where G is the base at the 5'-terminus. These results support the stacking model of camptothecin (Jaxel et al. (1991) J. Biol. Chem. 266, 20418-20423). Comparison of calf thymus and wheat germ topoisomerase I-mediated cleavage sites in the presence of camptothecin shows that many major cleavage sites are similar. However, the relative intensities are often different. One of the differences was attributable to a bias at position -3 where calf thymus topoisomerase I prefers G and wheat germ topoisomerase I prefers C. This difference may explain the unique patterns of cleavage sites induced by the two enzymes. Sequencing analysis of camptothecin-stimulated cleavage sites in the surrounding regions of point mutations in topoisomerase I cDNA, which were found in camptothecin-resistant cell lines, reveals no direct relationship between DNA cleavage sites in vitro and mutation sites.     

Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA.

Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.     

Induction of reversible complexes between eukaryotic DNA topoisomerase I and DNA-containing oxidative base damages. 7, 8-dihydro-8-oxoguanine and 5-hydroxycytosine

We recently showed that abasic sites, uracil mismatches, nicks, and gaps can trap DNA topoisomerase I (top1) when these lesions are introduced in the vicinity of a top1 cleavage site (Pourquier, P., Ueng, L.-M., Kohlhagen, G., Mazumder, A., Gupta, M., Kohn, K. W., and Pommier, Y. (1997) J. Biol. Chem. 272, 7792-7796; Pourquier, P., Pilon, A. A., Kohlhagen, G., Mazumder, A., Sharma, A., and Pommier, Y. (1997) J. Biol. Chem. 26441-26447). In this study, we investigated the effects on top1 of an abundant base damage generated by various oxidative stresses: 7,8-dihydro-8-oxoguanine (8-oxoG). Using purified eukaryotic top1 and oligonucleotides containing the 8-oxoG modification, we found a 3-7-fold increase in top1-mediated DNA cleavage when 8-oxoG was present at the +1 or +2 position relative to the cleavage site. Another oxidative lesion, 5-hydroxycytosine, also enhanced top1 cleavage by 2-fold when incorporated at the +1 position of the scissile strand. 8-oxoG at the +1 position enhanced noncovalent top1 DNA binding and had no detectable effect on DNA religation or on the incision step. top1 trapping by 8-oxoG was markedly enhanced when asparagine adjacent to the catalytic tyrosine was mutated to histidine, suggesting a direct interaction between this residue and the DNA major groove immediately downstream from the top1 cleavage site. Altogether, these results demonstrate that oxidative base lesions can increase top1 binding to DNA and induce top1 cleavage complexes.     

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.     

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.     

Base mutation analysis of topoisomerase II-idarubicin-DNA ternary complex formation. Evidence for enzyme subunit cooperativity in DNA cleavage.

Antitumor drugs, such as anthracyclines, interfere with mammalian DNA topoisomerase II by forming a ternary complex, DNA-drug-enzyme, in which DNA strands are cleaved and covalently linked to the enzyme. In this work, a synthetic 36-bp DNA oligomer derived from SV40 and mutated variants were used to determine the effects of base mutations on DNA cleavage levels produced by murine topoisomerase II with and without idarubicin. Although site competition could affect cleavage levels, mutation effects were rather similar among several cleavage sites. The major sequence determinants of topoisomerase II DNA cleavage without drugs are up to five base pairs apart from the strand cut, suggesting that DNA protein contacts involving these bases are particularly critical for DNA site recognition. Cleavage sites with adenines at positions -1 were detected without idarubicin only under conditions favouring enzyme binding to DNA, showing that these sites are low affinity sites for topoisomerase II DNA cleavage and/or binding. Moreover, the results indicated that the sequence 5'-(A)TA/(A)-3' (the slash indicates the cleaved bond, parenthesis indicate conditioned preference) from -3 to +1 positions constitutes the complete base sequence preferred by anthracyclines. An important finding was that mutations that improve the fit to the above consensus on one strand can also increase cleavage on the opposite strand, suggesting that a drug molecule may effectively interact with one enzyme subunit only and trap the whole dimeric enzyme. These findings documented that DNA recognition by topoisomerase II may occur at one or the other strand, and not necessarily at both of them, and that the two subunits can act cooperatively to cleave a double helix.     

DNA topoisomerase I cleavage sites in DNA and in nucleoprotein complexes.

The intracellular substrate for eukaryotic DNA topoisomerases is chromatin rather than protein-free DNA. Yet, little is known about the action of topoisomerases on chromatin-associated DNA. We have analyzed to what extent the organization of DNA in chromatin influences the accessibility of DNA molecules for topoisomerase I cleavage in vitro. Using potassium dodecyl sulfate precipitation (Trask et al., 1984), we found that DNA in chromatin is cleaved by the enzyme with somewhat reduced efficiency compared to protein-free DNA. Furthermore, using native SV40 chromatin and mononucleosomes assembled in vitro, we show that DNA bound to histone octamer complexes is cleaved by topoisomerase I and that the cleavage sites as well as their overall distribution are identical in histone-bound and in protein-free DNA molecules.     

The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I

Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.     

Spatial organization of topoisomerase I-mediated DNA cleavage induced by camptothecin-oligonucleotide conjugates

Triple helix-forming oligonucleotides covalently linked to topoisomerase I inhibitors, in particular the antitumor agent camptothecin, trigger topoisomerase I-mediated DNA cleavage selectively in the proximity of the binding site of the oligonucleotide vector. In the present study, we have performed a systematic analysis of the DNA cleavage efficiency as a function of the positioning of the camptothecin derivative, either on the 3′ or the 5′ side of the triplex, and the location of the cleavage site. A previously identified cleavage site was inserted at different positions within two triplex site-containing 59 bp duplexes. Sequence-specific DNA cleavage by topoisomerase I occurs only with triplex conjugates bearing the inhibitor at the 3′-end of the oligonucleotide and on the oligopyrimidine strand of the duplex. The lack of targeted cleavage on the 5′ side is attributed to the structural differences of the 3′ and 5′ duplex–triplex DNA junctions. The changes induced in the double helix by the triple-helical structure interfere with the action of the enzyme according to a preferred spatial organization. Camptothecin conjugates of oligonucleotides provide efficient tools to probe the organization of the topoisomerase I–DNA complex and will be useful to understand the functioning of topoisomerase I in living cells.     

The conformation of constitutive DNA interaction sites for eukaryotic DNA topoisomerase I on intrinsically curved DNAs.

The analysis of the sites which are cleaved constitutively and preferentially by eukaryotic DNA topoisomerase I on two intrinsically curved DNAs reveals the conformational features that provoke the cleavage reaction on the curve-inducing sequence elements in the absence of supercoiling. This analysis is based on the observation (Caserta et al. (1989) Nucleic Acids Res. 17, 8521-8532 and (1990) Biochemistry 29, 8152-8157) that the reaction of eukaryotic DNA topoisomerase I occurs on two types of DNA sites: sites S (Supercoiled induced) and sites C (Constitutive, whose presence is topology-independent). We report that sites C are abundant on the intrinsically curved DNAs analyzed. The DNAs studied were two intrinsically curved segments of different origin: the Crithidia fasciculata kinetoplast DNA and the bent-containing domain B of the Saccharomyces cerevisiae ARS1. On these DNA segments DNA topoisomerase I cleaves at the junctions between the poly(A) tracts and mixed-sequence DNA. Analysis of the conformation of the double helix around the cleavage sites has revealed that the reaction occurs in correspondence of a defined DNA conformational motif. This motif is described by the set of Eulerian angular values that define the axial path of DNA (helical twist, deflection angle, direction) and of the orthogonal components of wedge (roll and tilt).     

Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide).

Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.     

Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage

We have studied the effect of the antitumor drug, camptothecin, on the interaction of human topoisomerase I with DNA at the sequence level. At a low molar ratio of enzyme to DNA, cleavage is prominent and unique, located at a previously described hexadecameric recognition sequence, while a number of strong additional cleavage sites appear in the presence of the drug. Camptothecin stimulates cleavage at the recognition sequence less than twofold, whereas cleavage at the additional sites is stimulated up to 200-fold. Camptothecin greatly enhances the stability of the cleavable complexes formed at the additional sites, whereas the complex formed at the hexadecameric sequence is only marginally affected. Cleavage was eliminated at certain sites in the presence of camptothecin. Taken together these observations demonstrate that at least three types of potential eukaryotic topoisomerase I cleavage sites can be distinguished by the use of camptothecin. Comparison of the sequences at the additional cleavage sites in the presence of camptothecin reveals that the most frequently cleaved dinucleotide is TG with no consensus for the flanking nucleotides.     

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.     

Simian virus 40 minichromosomes contain torsionally strained DNA molecules.

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.     

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.     

DNA topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus with specific DNA cleavage activity

We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was capable of relaxing negatively supercoiled DNA at 75 degrees C in the presence of Mg2+. Mutation of the putative active site Tyr318 to Phe318 led to the inactivation of the protein. The S. solfataricus enzyme cleaved oligonucleotides in a sequence-specific fashion. The cleavage occurred only in the presence of a divalent cation, preferably Mg2+. The cofactor requirement of the enzyme was partially satisfied by Cu2+, Co2+, Mn2+, Ca2+, or Ni2+. It appears that the enzyme is active with a broader spectrum of metal cofactors in DNA cleavage than in DNA relaxation (Mg2+ and Ca2+). The enzyme-catalyzed oligonucleotide cleavage required at least 7 bases upstream and 2 bases downstream of the cleavage site. Analysis of cleavage by the S. solfataricus enzyme on a set of oligonucleotides revealed a consensus cleavage sequence of the enzyme: 5'-G(A/T)CA(T)AG(T)G(A)X / XX-3'. This sequence bears more resemblance to the preferred cleavage sites of topoisomerases III than to those of topoisomerases I. Based on these data and sequence analysis, we designate the enzyme S. solfataricus topoisomerase III.