Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage A 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 Agtll 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.Eukaryotic DNA topoisomerase II is a ubiquitous ATPdependent type II topoisomerase (reviewed in refs. [1][2][3]

ABSTRACTcDNA clones of the human TOP) gene encoding DNA topoisomerase I (EC 5.99.1.2) have been obtained by immunochemical screening of phage A libraries expressing human cDNA segments, using rabbit antibodies raised against purified HeLa DNA topoisomerase I. Hybridization patterns between the cloned cDNA sequences and human cellular DNA and cytoplasmic mRNAs indicate that human TOP) is a single-copy gene. The chromosomal location of the gene has been mapped to the long arm of chromosome 20, in the region q12-13.2, by hybridization of a radioactively labeled TOP1 cDNA probe to human metaphase chromosomes and to a panel of rodent-human somatic hybrids retaining overlapping subsets of human chromosomes.Eukaryotic DNA topoisomerase I (EC 5.99.1.2) was identified in extracts of mouse cells in 1972 (1) and has since been found in all eukaryotes (for recent reviews, see refs. 2-4). The enzyme catalyzes interconversions between different topological forms of DNA by transiently breaking DNA strands one at a time; it is therefore classified as a type I DNA topoisomerase (5).It has been known for many years that the eukaryotic enzyme is distinct from prokaryotic DNA topoisomerase I in terms of its substrate specificity and mechanism of catalysis.Recent sequencing of the TOP) gene encoding the enzyme in two distantly related yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe (6, 7), shows that the primary structure of the enzyme from the two organisms is closely related; sequence similarity is also found between the yeast enzymes and a type I DNA topoisomerase encoded by vaccinia virus (8). When the amino acid sequence of S. cerevisiae topoisomerase I is compared with that of Escherichia coli DNA topoisomerase I, however, no significant similarity is found (9). In spite of this lack of similarity, the expression ofactive yeast DNA topoisomerase I in E. coli has been shown to functionally complement the lethal phenotype of a mutation in the topA gene encoding the E. coli enzyme (10). These results indicate that the biological functions of eukaryotic and bacterial DNA topoisomerase I are a manifestation of their common denominator, namely, their catalysis of DNA topoisomerization.Eukaryotic DNA topoisomerase I appears to participate in a number of vital cellular processes including replication and transcription (2-4; [11][12][13][14] MATERIALS AND METHODS Materials. HeLa DNA topoisomerase I and rabbit antisera specific to the enzyme were prepared as described (14). A HeLa cDNA library in phage AgtlO was kindly provided by R. Tjian (University of California at Berkeley). Isolation, propagation, and characterization of human and mouse parental cell lines and somatic cell hybrids used in this work as well as the extraction of DNA from these cells were done as described previously (28,29). Peripheral blood from healthy volunteers was used in the preparation of metaphase chromosomes. All other materials were obtained from commercial sources.Methods. Immunochemical screening for the expression of human DNA topoisomeras...