Human Top1 Research Articles

A Camptothecin-resistant DNA Topoisomerase I Mutant Exhibits Altered Sensitivities to Other DNA Topoisomerase Poisons

The cytotoxic plant alkaloid camptothecin promotes DNA topoisomerase I-linked nicks in DNA by stabilizing a covalently bound enzyme-DNA complex. In the yeast Saccharomyces cerevisiae, substitution of Arg and Ala for the amino acid residues immediately N-terminal to the active site tyrosine in the yeast and human DNA topoisomerase I mutants, top1 vac, results in camptothecin resistance. To examine the mechanism of drug resistance, we assessed the sensitivity of these enzymes to several classes of DNA topoisomerase poisons. Yeast cells expressing the camptothecin-resistant top1 vac mutants were resistant to all of the camptothecin derivatives cytotoxic to wild-type TOP1-expressing cells. This correlated with a significant reduction in drug-induced DNA cleavage in vitro. However, the yeast and human mutant enzymes differed in their responses to the minor groove binding ligand netropsin and to saintopin, a DNA intercalator that targets both DNA topoisomerase I and II. The yeast mutant enzyme demonstrated enhanced sensitivity to the action of saintopin but was resistant to the inhibitory effects of netropsin. In contrast, the human Top1 vac enzyme was resistant to saintopin and indistinguishable from the wild-type enzyme in its response to the netropsin. These results are discussed in terms of enzyme function and the different modes of action of these DNA topoisomerase poisons.

Mouse genes encoding DNA topoisomerase I

We have initiated a genetic analysis of the physiologically important enzyme type I DNA topoisomerase in mouse. The exon-intron structures of the 5' part and the 3' part of the active gene, Top-1, were determined and shown to be quite similar to those of the previously determined human gene TOP1. The active mouse gene was mapped to the distal Chromosome (Chr) 2. In addition, the mouse genome contains one truncated processed topoisomerase-I-related pseudogene (retroposon), Top-1ps, on Chr 16. The Top-1ps locus, together with the immunoglobulin-lambda-light-chain locus, defines an additional conserved linkage group common to murine Chr 16 and human Chr 22, the site of the human pseudogene TOP1P2. The mapping data suggest that the pseudogene was established before mammalian radiation. Structural features, shared by the mouse and the human pseudogene, support this possibility.

YeastSaccharomyces cerevisiae as a model system to study the cytotoxic activity of the antitumor drug camptothecin

Eukaryotic DNA topoisomerase I catalyzes the relaxation of positively and negatively supercoiled DNA and plays a critical role in processes involving DNA, such as DNA replication, transcription and recombination. The enzyme is encoded by the TOP1 gene and is highly conserved in its amino acid sequence and sensitivity to the anti-neoplatic agent camptothecin. This plant alkaloid specifically targets DNA topoisomerase I by reversibly stabilizing the covalent enzyme-DNA intermediate. Presumably, it is the interaction of these drug-stabilized adducts with other cellular components, such as replication forks, that actually produces the DNA lesions leading to cell death. A conservation of the mechanism(s) of camptothecin-induced cell killing is also implicit in studies of the yeast Saccharomyces cerevisiae, where the camptothecin sensitivity of delta TOP1 yeast cells can be restored by plasmids expressing either yeast or human TOP1 sequences. This genetically tractable system is currently being exploited to describe the specific molecular interactions required for the cytotoxic action of camptothecin. The results of mutational analyses of yeast and human DNA topoisomerase I are presented, as well as a genetic screen designed to identify genes, other than TOP1, that are required for the cytotoxic activity of camptothecin.

Human DNA topoisomerase I is encoded by a single-copy gene that maps to chromosome region 20q12-13.2.

cDNA clones of the human TOP1 gene encoding DNA topoisomerase I (EC 5.99.1.2) have been obtained by immunochemical screening of phage lambda 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 TOP1 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.