Human TOP3: a single-copy gene encoding DNA topoisomerase III.

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.     

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.     

Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020.     

SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage

Ubiquitin/26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage. In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1/Smt3p, a ubiquitin-like protein. This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1/Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells. (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1/Smt3p. (iii) TOP1 physically interacts with UBC9. (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9/SUMO-1 may be involved in the repair of TOP1-mediated DNA damage.     

Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I.

Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA. Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond. Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme. From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively. Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue. There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.     

The t(11;20)(p15;q11) chromosomal translocation associated with therapy-related myelodysplastic syndrome results in an NUP98-TOP1 fusion.

The NUP98 gene is involved in 3 distinct chromosomal rearrangements, t(7;11)(p15;p15), t(2;11)(q31;p15), and inv(11)(p15q22); all of these NUP98 rearrangements have been identified in the malignant cells of patients with therapy-related acute myelogenous leukemia or myelodysplastic syndrome (t-AML/MDS). Here we report the cloning and characterization of a t(11;20)(p15;q11) translocation from patients with t-MDS. The breakpoint on chromosome 11p15 targets the NUP98 gene and results in the separation of the N-terminal FXFG repeats from the RNA-binding domain located in the C-terminus. The breakpoint on chromosome 20q11 occurs within the gene encoding human DNA topoisomerase I (TOP1). As a result, a chimeric mRNA encoding the NUP98 FXFG repeats fused to the body of DNA topoisomerase I is produced. These results indicate that NUP98 is a recurrent target in therapy-related malignancies, and that TOP1 is a previously unrecognized target for chromosomal translocations.     

DNA topoisomerase-targeting antitumor drugs can be studied in yeast.

The antitumor drugs camptothecin and an anilinoacridine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), which act on DNA topoisomerase I and II, respectively, are shown to inhibit the growth of Saccharomyces cerevisiae mutants selected for their permeability to other inhibitors. In addition to growth inhibition, these drugs induce high levels of homologous recombination and induce the expression of a DNA damage-inducible gene DIN3. Cytotoxicity of the drugs is more pronounced in strains that also carry a rad52 mutation. An analog of mAMSA), which is ineffective as an inhibitor of DNA topoisomerase II in mammalian cells, is also ineffective in eliciting physiological responses in these yeast strains. The physiological effects of camptothecin, but not those of mAMSA, disappear if the TOP1 gene encoding DNA topoisomerase I is disrupted. This shows that DNA topoisomerase I is the sole target of camptothecin cytotoxicity and illustrates that a nonessential enzyme can nevertheless be the target for a cytotoxic drug.     

cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment.

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.     

Expression of yeast DNA topoisomerase I can complement a conditional-lethal DNA topoisomerase I mutation in Escherichia coli.

We show that, despite differences in primary structure, substrate preference, and mechanism of catalysis, yeast DNA topoisomerase I can functionally substitute for Escherichia coli DNA topoisomerase I. A family of plasmids expressing the yeast TOP1 gene or 5'-deletion mutations of it were used to complement the temperature-sensitive phenotype of an E. coli topA mutant. These plasmids were then isolated from the cells by a rapid lysis procedure and examined for their degrees of supercoiling. Functional complementation of a conditional-lethal mutation in topA, which encodes E. coli DNA topoisomerase I, correlates with the expression of a catalytically active yeast enzyme that reduces the degree of negative supercoiling of intracellular DNA. We also show that approximately 130 amino acids of the amino-terminal portion of the yeast enzyme can be deleted without affecting its activity in vitro; activity of the enzyme inside E. coli, however, is more sensitive to such deletions.     

Effects of yeast DNA topoisomerase III on telomere structure.

The yeast TOP3 gene, encoding DNA topoisomerase III, and EST1 gene, encoding a putative telomerase, are shown to be abutted head-to-head on chromosome XII, with the two initiation codons separated by 258 bp. This arrangement suggests that the two genes might share common upstream regulatory sequences and that their products might be functionally related. A comparison of isogenic pairs of yeast TOP3+ and delta top3 strains indicates that the G1-3T repetitive sequence tracks in delta top3 cells are significantly shortened, by about 150 bp. Cells lacking topoisomerase III also show a much higher sequence fluidity in the subtelomeric regions. In delta top3 cells, clusters of two or more copies of tandemly arranged Y' elements have a high tendency of disappearing due to the loss or dispersion of the elements; similarly, a URA3 marker embedded in a Y' element close to the chromosomal tip shows a much higher rate of being lost relative to that in TOP3+ cells. These results suggest that yeast DNA topoisomerase III might affect telomere stability, and plausible mechanisms are discussed.     

DNA topoisomerase I drugs and radiotherapy for lung cancer

Lung cancer represents the most common cause of cancer-related mortality in the United States and around the world. DNA topoisomerase I (TOP1) drugs such as irinotecan and topotecan represent a unique class of chemotherapeutic agents that exhibit not only potent cytotoxic effect, but also tumor-selective radiation-sensitizing effect. The mechanism of cytotoxicity and radiation sensitization by TOP1 drugs has been intensely investigated. Modern radiotherapy, aided by improved imaging and treatment delivery technology, is capable of targeting tumors more precisely, while sparing surrounding critical structures. Clinical trials with camptothecin derivatives and radiotherapy have been conducted in lung cancers. Combined modality therapy with TOP1 drugs and radiotherapy offers a new frontier for lung cancer therapy. We review the present state of TOP1-targeted chemotherapy and modern radiotherapy for lung cancer.     

Acidic pH induces topoisomerase II-mediated DNA damage

Acidic pH plays an important role in various pathophysiological states and has been demonstrated to be carcinogenic in animal models. Recent studies have also implicated acidic pH in the development of preneoplastic Barrett's esophagus in human. However, little is known about the molecular mechanism underlying acidic pH-induced carcinogenesis. In the current study, we show that acidic pH, like the topoisomerase II (TOP2) poison VP-16 (demethylepipodophyllotoxin ethylidene-beta-D-glucoside), induces tumors in 9,10-dimethyl-1,2-benzanthracene(DMBA)-initiated mice. The following studies in tissue culture models have suggested that acidic pH acts like a TOP2 poison to induce TOP2-mediated DNA damage: (i) acidic pH induces TOP2-dependent DNA damage signals as evidenced by up-regulation of p53 and Ser-139 phosphorylation of H2AX [a substrate for ataxia telangiectasia mutated (ATM)ATM and Rad3-related (ATR) kinases]; (ii) acidic pH-induced cytotoxicity in tumor cells is reduced in TOP2-deficient cells; (iii) acidic pH increases the mutation frequency of the hypoxanthine phosphoribosyl transferase (HPRT) gene in a TOP2-dependent manner; and (iv) acidic pH induces reversible TOP2-mediated DNA strand breaks in vitro. We discuss the possibility that TOP2-mediated DNA damage may contribute to acidic pH-induced carcinogenesis.     

Topoisomerase I identified by scleroderma 70 antisera: enrichment of topoisomerase I at the centromere in mouse mitotic cells before anaphase.

Antibodies derived from scleroderma patients positive for the extractable 70-kDa antigen were shown to react with topoisomerase I. Topoisomerase I was identified by molecular size and by antibody inhibition of the topoisomerase I-specific relaxation of supercoiled plasmid DNA. By using in situ localization by indirect fluorescence, we found topoisomerase I preferentially located in the centromeric regions of the mouse G2-phase cells and chromosomes, while the distribution in human cells is much more dispersed. Moreover, comparison of a published consensus sequence for topoisomerase I binding with mouse satellite DNA revealed a high degree of homology. The localization of topoisomerase I in the centromeres of mouse cells in the later part of the cell cycle and prior to anaphase suggests functional involvement in mitosis.     

Isolation and characterization of the gene encoding Drosophila DNA topoisomerase II.

We have isolated the gene coding for the Drosophila type II DNA topoisomerase by immunochemically screening a Drosophila cDNA library constructed with a phage lambda expression vector, lambda gt11. The identity of the cloned gene is confirmed by the analysis of an antigenic fusion protein produced in Escherichia coli and by the in vitro translation of its RNA. The gene is 5.1 kilobases in length, the expected size for a gene encoding topoisomerase II (Mr 170,000), and it is divided into five exons. By in situ hybridization to the polytene chromosomes from salivary glands, we have mapped it to chromosome 2L at 37D.     

The alpha-spectrin gene is on chromosome 1 in mouse and man.

By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies.     

Chromosomal localization of human β globin gene on human chromosome 11 in somatic cell hybrids

We have successfully used a DNA.cDNA molecular hybridization assay to directly determine the presence or absence of human beta globin gene sequences in 20 human-mouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human beta globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human beta globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human beta globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT-4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT-4. 157-BNPT-4 contained the human beta globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human gamma globin cDNA; two lines positive for human beta globin gene sequences also contained human gamma globin gene sequences while one line was negative for both beta and gamma gene sequences. On the basis of these results, the human beta and gamma globin genes have been assigned to human chromosome 11.