Topoisomerases are ubiquitous enzymes that play critical roles in the read-out and replication of the genome. All members of this superfamily of enzymes work by breaking DNA, permitting the strands to achieve new topological relations to one another, and finally sealing the break. The enzyme cycle contains an intermediate in which the protein is covalently linked to the DNA via a high energy bond. Murphy?s Law predicts, and experiments bear out, that this cycle of breakage and reunion will occasionally fail. When this happens the topoisomerase becomes covalently trapped at a broken end of DNA. Such discontinuities are dangerous. For a dividing cell the alternatives are either to fix the break or to suffer one of the following fates: permanent arrest of the cell cycle, apoptosis, or fragmentation of the genome. These disastrous occurrences can only be prevented by timely and faithful repair. There are many well-characterized mechanisms within a cell to repair broken DNA but topoisomerase-linked breaks present a special problem because the end of the break is encumbered with covalently joined protein residues. Successful repair must involve the removal of these residues from the DNA; in fact, it seems logical to imagine that such removal would be one of the first steps in repair. Surprisingly, there has been very little research on the removal of convalently-linked topoisomerase from the broken ends of DNA. This project focuses on such removal of one particular class of enzyme, eukaryotic topoisomerase I. Our entry into this field was the serendipitous discovery of an enzymatic activity that specifically hydrolyzes the bond connecting a tyrosine to 3?-end of DNA, i.e., the bond that comprises the topoisomerase I covalent link. In previous work we cloned the gene encoding the enzyme (which we named TDP1) and showed that it was highly conserved in eukaryotes from yeast to human. We also found that the yeast gene played an important role in repair of lesions induced by a toxic topoisomerase, although repair of lesions induced by the drug camptothecin depended less on TDP1 function. Our present work addresses three concerns. First, on the expectation that ?spontaneous? topoisomerase damage occurs during normal growth, we have focused on the role of the TDP1 gene in the absence of an artificial source of topoisomerase damage. A survey of various parameters has turned up only one significant alteration in a tdp1 mutant: spontaneous mutations (measured at the CAN1 locus) are modestly but significantly increased. Most importantly, this increase is not observed in a strain lacking topoisomerase 1. Second, we remain aware that yeast is a model system and the ultimate impact of our work on repair of topoisomerase damage will undoubtedly come from applications to higher eukaryotes. To expedite such studies we prepared a clone of the full-length human cDNA for TDP1, generated an antibody to the bacterially-expressed human enzyme, and have worked out a Western blot assay that detects Tdp1 in extracts of human cells. Third, we are seeking to identify those enzymes in yeast that are responsible for unlinking topoisomerase 1 from DNA in the pathway(s) that function in parallel to the TDP1-dependent pathway. We used reverse genetics to evaluate three plausible candidates, APN1, RAD1, and the nuclease function of MRE11. None of these proved to have a major effect on survival of topoisomerase damage. Rather than continue the serial evaluation of plausible candidates, we have embarked on a forward random mutagenesis for genes that work in parallel with TDP1. Specifically, we have chemically mutagenized a tdp rad1 erg6 strand (the latter mutation needed for camptothecin entry) and screened colonies by replica plating for camptothecin hypersensitivity. We focused on that subset of mutants whose sensitivity decreased when a plasmid-born copy of TDP1 was transformed into the cell. Thus, we are assembling a collection of mutations that enhance the tdp1 phenotype.
| 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 |
