Distribution of topoisomerase II cleavage sites in simian virus 40 DNA and the effects of drugs.

The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site.     

Chromosomal mapping and nucleotide sequence of a human DNA autonomously replicating sequence

A 1.1-kb human DNA fragment (ARSH1) capable of functioning as a putative origin of replication in yeast cells has been characterized both by in situ hybridization to human metaphase chromosomes and by DNA sequencing. Our hybridization studies show a preferential localization of ARSH1 in chromosome regions 1p34-36 and 2q34-37. DNA sequence analysis indicates that in addition to the consensus sequence required for ARS function in yeast cells, nuclear matrix-associated DNA motifs are also present in the 1.1-kb fragment. These results suggest that ARSH1 sequences may serve as points of anchorage to the nuclear matrix for chromosomes 1 and 2.     

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.     

Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts

A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.     

Preferential, cooperative binding of DNA topoisomerase II to scaffold-associated regions.

DNA elements termed scaffold-associated regions (SARs) are AT-rich stretches of several hundred base pairs which are known to bind specifically to nuclear or metaphase scaffolds and are proposed to specify the base of chromatin loops. SARs contain sequences homologous to the DNA topoisomerase II cleavage consensus and this enzyme is known to be the major structural component of the mitotic chromosome scaffold. We find that purified topoisomerase II preferentially binds and aggregates SAR-containing DNA. This interaction is highly cooperative and, with increasing concentrations of topoisomerase II, the protein titrates quantitatively first SAR-containing DNA and then non-SAR DNA. About one topoisomerase II dimer is bound per 200 bp of DNA. SARs exhibit a Circe effect; they promote in cis topoisomerase II-mediated double-strand cleavage in SAR-containing DNA fragments. The AT-rich SARs contain several oligo(dA).oligo(dT) tracts which determine their protein-binding specificity. Distamycin, which is known to interact highly selectively with runs of A.T base pairs, abolishes the specific interaction of SARs with topoisomerase II, and the homopolymer oligo(dA).oligo(dT) is, above a critical length of 240 bp, a highly specific artificial SAR. These results support the notion of an involvement of SARs and topoisomerase II in chromosome structure.     

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.     

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.     

Analysis of the chicken DNA fragments that contain structural sites of attachment to the nuclear matrix: DNA-matrix interactions and replication

Ten short DNA fragments have been selected from a library of the nuclear matrix-attached DNA (nmDNA) from chicken erythrocytes by their ability to hybridize with the fraction of chicken replication origins isolated by nascent DNA strand extrusion. The primary structure of these fragments has been determined. Five of the sequences contained a topoisomerase II recognition site. Most of the studied DNA fragments also have a common eight-nucleotide motif, GCAGACCG/A. A sequence-specific DNA-binding protein with a MW of 55 kDa that interacted with this motif has been identified. Some of the cloned DNA fragments promoted an increased level of transient plasmid replication in transfected chicken cells. The ability of plasmid bearing nmDNA fragments to replicate correlated directly with their ability to target plasmids to the nuclear matrix compartment.     

The ATP-operated clamp of human DNA topoisomerase IIalpha: hyperstimulation of ATPase by "piggy-back" binding

We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction.     

Topoisomerase IIB and an extracellular nuclease interact to digest sperm DNA in an apoptotic-like manner

We previously demonstrated that mammalian spermatozoa contain a nuclease activity that cleaves DNA into loop-sized fragments. We show here that this activity is mediated by a nuclear matrix-associated topoisomerase IIB (TOP2B) interacting with an extracellular Mn2+/Ca2+-dependent nuclease. Together, these enzymes cleave all of the DNA into fragments of 50 kb, and this cleavage can be reversed by EDTA. If dithiothreitol is included, the nuclease digests the DNA, and if the protamines are removed the DNA is completely digested. A similar, TOP2B-mediated, chromatin fragmentation, which is reversible, followed by digestion of the DNA by an intracellular nuclease occurs in somatic cells during apoptosis. The extracellular location of the sperm nuclease made it possible to reconstitute the fragmentation activity in isolated spermatozoa, thus allowing us to identify two novel aspects of the mechanism. First, the fragmentation of all of the DNA to 50 kb by TOP2B required the addition of the extracellular nuclease or factor. Second, the subsequent, complete digestion of the DNA by the nuclease could be inhibited by etoposide, suggesting that the nuclease digestion requires TOP2B religation of the cleaved DNA. These data are the first demonstration of an active TOP2B in spermatozoa, suggesting this inert chromatin may be more active than previously thought. They also show that the unique chromatin structure of spermatozoa may provide an important model to study the regulated degradation of chromatin by TOP2B and associated nucleases.     

Interaction between yeast sgs1 helicase and DNA topoisomerase III

The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases that includes the human Bloom's syndrome and Werner's syndrome proteins. In this work, we report studies on the interaction between Sgs1 and DNA topoisomerase III in vitro and in vivo. Affinity chromatography experiments with various fragments of Sgs1, a 1447-amino acid polypeptide, suggested that its N-terminal one-fifth was sufficient for interaction with DNA topoisomerase III. Gel electrophoretic mobility shift assays also indicated that a fragment Sgs1(1-283), containing residues 1-283, inhibited the binding of DNA topoisomerase III to single-stranded DNA. A shorter protein fragment containing residues 1-107 also showed partial inhibition in these assays. Studies of a sgs1 top1 double mutant lacking both Sgs1 and DNA topoisomerase I showed that the slow growth phenotype of this double mutant is suppressed by expressing full-length Sgs1, but not Sgs1 without the N-terminal 107 amino acid residues. In sgs1 top3 cells devoid of DNA topoisomerase III, however, expression of full-length Sgs1 or Sgs1 lacking the N-terminal 107 amino acid residues has the same effect of reducing the growth rate of the double mutant. These in vitro and in vivo data indicate that Sgs1 and DNA topoisomerase III physically interact and that this interaction is physiologically significant.     

Mammalian topoisomerase II activity is modulated by the DNA minor groove binder distamycin in simian virus 40 DNA

DNA topoisomerases II are nuclear enzymes that have been identified recently as targets for some of the most active anticancer drugs. Antitumor topoisomerase II inhibitors such as teniposide (VM-26) produce enzyme-induced DNA cleavage and inhibition of enzyme activity. By adding to such reactions distamycin, a compound whose effects on DNA have been extensively characterized, we investigated the effects of drug binding upon topoisomerase II-mediated DNA cleavage induced by VM-26. We have found a correspondence between distamycin binding (determined by footprinting analysis) and topoisomerase II-mediated cleavage of SV40 DNA (determined by sequencing gel analysis). Distamycin binding potentiated the cleavage of specific sites in the near proximity of distamycin-binding sites (within at least 25 base pairs), which indicates that DNA secondary structure is involved in topoisomerase II-DNA interactions. That distamycin potentiated cleavage only at sites that were recognized in the absence of distamycin and suppressed cleavage directly at distamycin-binding sites indicates that topoisomerase II recognizes DNA on the basis of primary sequence. In addition, distamycin stimulated topoisomerase II-mediated DNA relaxation and antagonized the inhibitory effect of VM-26. These results show that the DNA sequence-specific binding of distamycin produces local and propagated effects in the DNA which markedly affect topoisomerase II activity.     

Topoisomerase inhibitors induce irreversible fragmentation of replicated DNA in concanavalin A stimulated splenocytes

Etoposide, a nonintercalative antitumor drug, is known to inhibit topoisomerase II. Its effects have been tested in concanavalin A stimulated splenocytes, a system of cell proliferation in which topoisomerase II is induced. The primary effect of etoposide was a strong inhibition of DNA synthesis and the production of reversible DNA breaks, presumably associated with topoisomerase II. However, prolonged (20 h) contact with the drug resulted in a secondary fragmentation by irreversible double-strand breaks that yielded unusually small DNA fragments. Surprisingly, the same effect was obtained with novobiocin, which does not produce topoisomerase II associated DNA breaks. Moreover, long-term treatment with camptothecin, a specific inhibitor of topoisomerase I which is known to induce single-strand breaks in vitro and in vivo, also produced double-strand breaks and DNA fragmentation into small pieces. These findings suggest that prolonged treatment of proliferating splenocytes by etoposide and other topoisomerase inhibitors induced DNA fragmentation by a mechanism that does not directly involve topoisomerases.     

Eukaryotic DNA topoisomerase I reaction is topology dependent.

The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism which encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.