Several years ago our laboratory reported that extracts of eukaryotic cells contain a novel phosphodiesterase activity, one that is capable of hydrolyzing a bond between tyrosine and the 3?-end of DNA. In nature, the only known occurrence of this type of linkage is in the covalent complex that is transiently formed between DNA and eukaryotic topoisomerase I (TopI). Accordingly, we suggested that the enzyme responsible for the novel activity was involved in repair of aborted Top1 intermediates. This prediction was at least partially borne out following our identification of a gene in Saccharomyces cerevisiae that encodes an enzyme with tyrosyl-DNA phosphodiesterase activity; null mutations of this gene (TDP1) increased the lethal effectiveness of camptothecin (CPT), a drug that specifically blocks the completion of the Top1 catalytic cycle. Interest in TDP1 has been growing. In large measure this is due to that fact that the gene is highly conserved in eukaryotes, with identifiable orthologs in a wide variety of animals, including man. Messenger RNA for TDP1 is found in many human tissues and this expression appears to have functional significance since a human disease is associated with the inheritance of a mutation in the gene. With an eye to understanding its relevance to all eukaryotes, our recent work has been focused on defining the biological niche for Tdp1 in budding yeast. Increased sensitivity to CPT was previously observed only when a tdp1 mutation was added to yeast strains that also carried mutations in other repair genes. This suggested that the pathway involving this enzyme is only one of several alternative pathways for dealing with genomic damage. If so, the importance of TDP1 should become more obvious when damage levels are high and alternative pathways become saturated. To test this hypothesis we constructed strains that overexpress TopI and thus raise the level of damage engendered by CPT. Under these circumstances, we indeed find that a substantial effect of TDP1 on survival even without the benefit of sensitizing mutations. But, if these highly engineered conditions were the only ones under which TDP1 were non-redundant, it is hard to imagine why the gene is conserved throughout evolution. Accordingly we searched for and found more natural cases in which the influence of the gene is apparent in the absence of accessory mutations. First, we assessed the acute response to Top1 damage. Specifically, wild-type and tdp1 mutant cells were fixed and DAPI stained during exposure to CPT and then examined by fluoresence microscopy for nuclear morphology. Within two hours of exposure the majority of wild-type cells had suffered cell cycle arrest. As expected from a previous report, the predominant morphological form was indicative of arrest at the G2/M boundary. But, with time there was also an increasing proportion of cells in which the nucleus was elongated and stretched through the bud neck, indicative of arrest in mid-anaphase. Cells bearing a deletion of TDP1 showed the same overall pattern but there was a significant quantitiative difference. At every time point tested, the proportion of mutant cells in mid-anaphase arrest was modestly but consistently higher than that for the wild-type strain. Thus, although survival tests show that cells can recover from CPT damage without Tdp1, the altered arrest distribution shows that mutant cells do not process checkpoint-inducing lesions with normal kinetics. This confirms and extends the conclusion that TDP1 is important for repair in cells undergoing high levels of Top1 damage. Is TDP1 conserved in the genome of eukaryotes only for its ability to repair extreme levels of damage or does the gene also serve a non-redundant daily housekeeping function? When no DNA damage is artificially induced and no alternative pathways for repair are inactivated, we have found that yeast strains bearing a tdp1 null mutation are roughly equivalent to control strains in their ability to grow on a variety of media and at various temperatures, ability to mate and sporulate, and ability to enter and exit stationary phase. In addition, FACS analysis of cells in the midst of exponential growth reveals no significant difference in cell cycle distribution. In fact, the only published report of an unconditional effect of a tdp1 mutation claimed a subtle alteration in nuclear structure and a modest defect in gene silencing. We constructed strains to follow up this observation but failed to observe an effect of a tdp1 mutation on nuclear morphology (using a NUP49-GFP fusion) or telomeric silencing (using a VRTEL::URA3 reporter). We also obtained the strains used in the original paper but could not reproduce the published effect of TDP1 on rDNA silencing. We suspect that our failures reflect the difficulty of assessing subtle changes with non-quantitative assays. This point of view is supported by our success in discovering a small but consistent effect of TDP1 on spontaneous mutagenesis, a process that is traditionally evaluated in a precise and quantitative manner. The rate of spontaneous mutatagenesis (the average number of new mutations introduced into the genome per generation) at the CAN1 locus arising during normal growth was evaluated fora set of isgenic strains by the method of the median. This showed that removal of TDP1 from a wild-type strain is associated with a significant increase in mutation rate. The average increase, although small, is comparable or larger than that reported for other repair genes, e.g., RAD1, RAD6, APN1. Two candidates for the source of these additional mutations are Top1 lesions and oxidative damage, each of which can occur spontaneously as accidental events: in either case, removal of Tdp1 would presumably result in a shift to modes of repair that are more prone to error. We favor Top1 lesions as the culprit as a result of our comparison of spontaneous mutation rates of top1 and top1 tdp1 strains. When introduced into a wild-type strain, a top1 mutation by itself had a mild mutagenic effect but there was no increase in mutagenesis rate when TDP1 was inactivated in this background. The simplest explanation of these data is that Tdp1 normally functions to repair spontaneous Top1 lesions. The view that emerges from our studies is a two-fold role for TDP1 in budding yeast. On the one hand, when the cell is confronted with a heavy burden of damage, the enzyme is involved in life or death decisions. On the other hand, at lower levels of damage, as judged by checkpoint responses and mutation rate, it is important for the quality of the
| 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 |
