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'.     

The basis for camptothecin enhancement of DNA breakage by eukaryotic topoisomerase I.

The eukaryotic topoisomerase I (topo I) is the target of the cytotoxic alkaloid camptothecin (CTT). In vitro, CTT enhances the breakage of DNA by topo I when the reaction is stopped with detergent. Although breakage at some sites is enhanced to a great extent while breakage at others is enhanced only minimally, CTT does not significantly change the breakage specificity of topo I in vitro. It has been suggested that CTT acts by slowing the reclosure step of the nicking-closing reaction. To test this hypothesis, we have measured the rate of reclosure for different break sites in the presence of CTT after adding 0.5 M NaCl to a standard low salt reaction. In support of the hypothesis, we find that topo I-mediated DNA breakage is enhanced the greatest at those sites where closure of the break is the slowest. These results suggest a mechanism for the toxicity of CTT in vivo.     

Histone H1 inhibits eukaryotic DNA topoisomerase I.

Histone H1 inhibits the catalytic activity of topoisomerase I in vitro. The relaxation activity of the enzyme is partially inhibited at a molar ratio of one histone H1 molecule per 40 base pairs (bp) of DNA and completely inhibited at a molar ratio of one histone H1 molecule per 10 base pairs of DNA. Increasing the amount of enzyme at a constant histone H1 to DNA ratio antagonizes the inhibition. This indicates that topoisomerase I and histone H1 compete for binding sites on the substrate DNA molecules. Consistent with this we show on the sequence level that histone H1 inhibits the cleavage reaction of topoisomerase I on linear DNA fragments.     

Association of eukaryotic DNA topoisomerase I with nucleosomes and chromosomal proteins.

A DNA topoisomerase activity is found to be associated with the nucleosomes released by the Staphylococcal nuclease digestion of HeLa nuclei. Such an association is found to be salt dependent. A number of criteria have established that this DNA topoisomerase activity is due to HeLa topo I (Liu, L. F. and Miller, K. G. (1980) Proc. Natl. Acad. Sci. USA 78, 3489-3491). A similar association has been demonstrated from the in vitro studies using purified mononucleosomes and eukaryotic DNA topoisomerase I. Nonhistone HMG proteins and histone H1 are found to stimulate topoisomerase activity in vitro and form tight complexes with eukaryotic DNA topoisomerase I. The intimate interactions of topoisomerase I with chromosomal proteins and nucleosomes may be an essential feature of the topoisomerase function in vivo.     

Quantitation of eukaryotic topoisomerase I reactivity with DNA. Preferential cleavage of supercoiled DNA

A method has been used to quantitate the reaction between eukaryotic type I DNA topoisomerase and topological forms of DNA. This procedure (Trask, D.K., DiDonato, J.D. and Muller, M.T. (1984) Eur. Mol. Biol. Organ. J. 3, 671-676) measures the efficiency of DNA cleavage and concurrent formation of a covalent enzyme/DNA complex. Eukaryotic type I topoisomerases react preferentially by 5-10-fold with supercoiled DNA. The effect of supercoiling is clearly evident in that both the initial rate and final extent of the reaction is elevated. Because the dissociation rate is much lower than the association rate, it is possible to isolate native topoisomerase/DNA complexes. These complexes are comprised of enzyme molecules which are catalytically active when challenged with a second supercoiled DNA substrate. Collectively, the data support the conclusion that a functional intermediate in the reaction sequence is being detected and that the avian topoisomerase I preferentially cleaves supercoiled DNA.     

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.     

Structural characterization of intrinsically curved AT-rich DNA sequences.

AT-rich DNA sequences other than AnTm tracts (n + m > or = 4) are known to be intrinsically curved. The AATAT-element constitutes one known example of these sequences. In this paper, the elucidation of the structural basis of the curvature induced by this sequence element was addressed. As judged by the patterns of cleavage by the hydroxyl radical and DNase I, the AATAT sequence shows a narrow minor groove. Furthermore, the 5' adenine residue of the AA dinucleotide contained within the sequence is hyperreactive to diethylpyrocarbonate. Similar structural properties are shown by several sequences inducing intrinsic DNA curvature, such as an A5-tract or the closely related ATAAT, AATATA and TAATAT sequences, which are also shown here to induce curvature. On the other hand, other related sequences, such as TATAA and ATATA, that do not induce curvature, show different structural characteristics.     

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.     

Mapping of the active site tyrosine of eukaryotic DNA topoisomerase I

DNA topoisomerase I from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe was overproduced using the cloned genes. Extracts from cells overproducing DNA topoisomerase I were prepared and incubated with 32P-labeled DNA. Alkali was used to trap the topoisomerase I-DNA covalent intermediate. Most of the DNA was digested with nuclease, and the resultant 32P-labeled topoisomerase I was subjected to cleavage with cyanogen bromide or formic acid. From the molecular weights of the resultant labeled peptides and by comparison of the amino acid sequences derived from the cloned genes, we were able to deduce that the active site tyrosine of eukaryotic DNA topoisomerase I is very near the carboxyl terminus, at amino acid 771 for S. pombe and 727 for S. cerevisiae. Site-directed mutagenesis was used to change tyrosine 727 of S. cerevisiae topoisomerase I to a phenylalanine. The resulting mutant topoisomerase I protein lost all DNA relaxation activity and rendered cells resistant to the topoisomerase I inhibitor, camptothecin. The amino acid sequence of human topoisomerase I has significant similarity to the two yeast topoisomerase I sequences. Based on this similarity, we infer that tyrosine 723 is the active site tyrosine of human enzyme.     

Simultaneous purification of DNA topoisomerase I and II from eukaryotic cells

We have developed a procedure for the simultaneous purification of DNA topoisomerase I and II from calf thymus. Both enzymes were first extracted from isolated nucleoprotein complexes. After batchwise chromatography on hydroxylapatite the two enzyme activities were separated on a FPLC phenylsuperose column. The enzymes were further purified by a second chromatography on phenylsepharose (topo I) or FPLC Mono Q (topo II). The purification can be finished within three days, yielding 0.5-1.0 mg quantities of homogeneous, enzymatic active preparations of the two proteins from 200 g of starting material.     

Camptothecin inhibits both the cleavage and religation reactions of eukaryotic DNA topoisomerase I.

We investigated the mode of action of the antitumor drug, camptothecin, by use of a partly double-stranded suicide DNA substrate which enables uncoupling of the cleavage and religation half-reactions of topoisomerase I. The suicide DNA substrate contains a single topoisomerase I site at which SDS cleavage is strongly enhanced by camptothecin on normal double-stranded DNA. The results show that the religation reaction of topoisomerase I per se is strongly inhibited at this site compared to site that is only marginally affected by camptothecin on double-stranded DNA. This study hereby directly demonstrates that camptothecin-mediated stability of a topoisomerase I-DNA complex is sequence-dependent. The influence of camptothecin on the suicide cleavage reaction of topoisomerase I was also investigated. Surprisingly, the cleavage reaction per se is strongly inhibited by the drug. However, reformation of a cleavable suicide DNA substrate, which is fully double-stranded downstream from the cleavage position except for a nick, completely reverses the inhibitory effect of the drug on the cleavage reaction. The results suggest that the inhibitory effect of camptothecin on cleavage is due to a general decrease in the noncovalent interaction of topoisomerase I with partly double-stranded suicide DNA substrates. Based on the findings, a plausible model for camptothecin action is discussed.     

Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence

The interaction between eukaryotic DNA topoisomerase I and a high affinity binding sequence was investigated. Quantitative footprint analysis demonstrated that the substrate preference results from strong specific binding of topoisomerase I to the sequence. The specificity was conferred by a tight noncovalent association between the enzyme and its target DNA, whereas the transient formation of a covalently bound enzyme.nicked DNA intermediate contributed insignificantly to the overall affinity. Topoisomerase I protected both strands over a 20-base pair region in which the cleavage site was centrally located. DNA modification interference analysis revealed a 16-base pair interference region on the scissile strand. Essential bases were confined to the 5' side of the cleavage site. The 6-base pair interference region observed on the complementary strand did not contain essential bases.     

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA.

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.     

Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.     

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.     

Negative supercoiling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate.

In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.     

The mechanism of the supercoiled DNA recognition by eukaryotic type I topoisomerases. I. The enzyme interaction with nonspecific oligonucleotides

Interaction of the DNA type I topoisomerases from the murine and human placenta cells with nonspecific oligonucleotides was analyzed. The contributions of strong and week nonspecific electrostatic, van der Waals's, and hydrophobic interactions, and hydrogen bonding of the enzymes to the complex formation with the single- and double-stranded DNAs were determined. The factors that determine the top-priority recognition of the topologically stressed DNA were revealed. The results were interpreted in comparison with the X-ray analysis data for human DNA topoisomerase I.