Topoisomerases are crucial cellular enzymes that work by breaking the DNA backbone, allowing or encouraging topological change, and resealing the break. The enzymes are efficient because DNA breakage is accompanied by covalent union between protein and DNA to create an intermediate that is resolved during the resealing step. This mechanism, although elegant, also makes topoisomerases potentially dangerous. If the resealing step fails, a normally transient break in DNA becomes a long-lived disruption, one with a topoisomerase protein covalently joined to it. Unless a way is found to restore the continuity of DNA, the cell will die. Repair of topoisomerase covalent complexes is of obvious value to the cell but the subject remains largely unexplored. A plausible pathway invokes hydrolysis of the bond joining the topoisomerase to DNA; release of the topoisomerase would then permit the cleaved DNA to undergo conventional modes of break repair. We previously described an activity that specifically hydrolyzes the type of bond found in complexes between DNA and topoisomerase I, a phosphodiester between the 3-end of DNA and the phenoxy moiety of tyrosine. We have now succeeded in cloning the gene for tyrosyl-DNA phosphodiesterase (TDP). From a library of yeast genomic fragments, we obtained a plasmid that corrected the hypersensitivity to the topoisomerase poison camptothecin (CPT) displayed by a strain of yeast with low TDP activity that had been previously isolated and characterized in our lab. Subcloning this fragment showed that the responsible ORF was YBR223c, a previously uncharacterized protein of 544 amino acids. To distinguish whether YBR223c encodes or controls TDP activity, we introduced a histidine- tagged version into Escherichia coli, which by itself has no detectable TDP activity. Induction of bacteria bearing this construct resulted in massive overproduction of TDP. We conclude that YBR223c encodes the enzyme and have accordingly renamed this gene TDP1. Database searches failed to reveal homology between TDP1 and any genes of known function. Even individualized comparisons to motifs identified in various phosphodiesterases and phosphatases were, at best, marginal. On this basis we conclude that TDP1 encodes a novel enzyme. However, eukaryotic (but not prokaryotic) databases contain several unannotated sequences that match TDP1, a finding consistent with the distribution of enzyme activity. In mouse and man there are several EST entries that can be aligned to make up a single ORF with substantial similarity to the carboxy-terminal half of TDP1. We used PCR of a human cDNA library to identify sequences from the 5 half of the gene and to construct a nearly full-length human cDNA. Overexpression of the latter in bacteria of leads to massive expression of TDP activity. We conclude that the TDP1 gene is highly conserved in eukaryotes. Isolation of the TDP1 gene will allow studies of the enzymology and cell biology of a new kind of DNA repair. The gene also provides a potential tool to improve chemotherapy with camptothecins and other topoisomerase I poisons. Although these are promising anti-cancer drugs, their value is often limited by resistance of tumor cells and/or sensitivity of non-tumor cells. Repair of the topoisomerase lesion is likely to be one of the factors that determine the level of cellular sensitivity to topoisomerase poisons. With the TDP1 gene in hand, one can readily assess the expression of this enzyme in individual patients and possibly predict the likelihood of therapeutic success. Moreover, if genetic technology can be used to alter levels of this enzyme, the efficacy and safety of the drugs may be improved. - topoisomerase, phosphodiesterase, S. cerevisiae, DNA repair
| Liu, Chunyan; Pouliot, Jeffrey J; Nash, Howard A (2004) The role of TDP1 from budding yeast in the repair of DNA damage. DNA Repair (Amst) 3:593-601 |
| Liu, Chunyan; Pouliot, Jeffrey J; Nash, Howard A (2002) Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1. Proc Natl Acad Sci U S A 99:14970-5 |
| Pouliot, J J; Robertson, C A; Nash, H A (2001) Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae. Genes Cells 6:677-87 |
