Our laboratory previously discovered a novel phosphodiesterase that is capable of removing non-nucleotide residues from the 3'-end of DNA. After intial biochemical characterization, we identified the gene that encodes such activity in Saccharomyces cerevisiae, named it TDP1, and showed that orthologs of this gene are present throughout the eukaryotic kingdom. Two residues that are known substrates for the yeast and human enzymes, 3'-tyrosine and 3'-phosophoglycolate (PG), suggest that Tdp1 is involved in repair of topoisomerase 1 (Top1) damage and oxidative damage. Indeed, we and others have shown that the addition of a tdp1 mutation to genetically sensitized yeast cells yields increased sensitivity to a Top1 poison, camptothecin (CPT), and an oxidative posion, bleomycin (BLM). In the past year we have answered several unresolved questions about Tdp1 function in repair. 1) Our previous studies used null or deletion mutations to assess the role of Tdp1. Such mutations both remove the source of enzymatic activity and preclude any possible participation of the protein in repair complexes. To judge the relative importance of these two deficits, we wanted to compare a TDP1 knockout with a point mutant that would leave expression of the gene intact but disable the active site. Work done by another lab had identified the active site nucleophile of the human enzyme; we accordingly engineered a His to Ala mutation into the corresponding residue of the yeast enzyme. Western blotting of crude extracts of mutant yeast showed that gene expression was only slightly reduced but biochemical assays of these extracts revealed no Tdp1 activity. The sensitivity to CPT and BLM of the new point mutant and the deletion mutant were compared in appropriately sensitized genetic backgrounds and found to be indistingushable from each other but notably increased when compared to strains bearing a wild-type TDP1 gene. We conclude that the principal contribution of Tdp1 to repair is via its enzymatic activity. 2) When purified from yeast, Tdp1 is about 50 times more active on tyrosine than on PG substrates, raising the possibility that TDP1 affects survival following BLM treatment not via direct action on PG residues but because oxidative damage leads to Top1 trapping. To test this possibility, we introduced a deletion of the TOP1 gene into a set of strains that were sensitized to BLM damage by inactivation of the APN1 and APN2 genes. The member of this set that carried a deletion of the TDP1 gene was clearly more sensitive than the member with a wild-type TDP1 gene. The difference was confirmed by an experiment in which a plasmid bearing a wild-type copy of the gene was added to the mutant strain. Having eliminated the hypothesis that Tdp1 contributes to survival after BLM only through its repair of trapped Top1, we now favor hypotheses that invoke hydrolysis of PG residues by Tdp1 as the critical factor. According to these hypotheses, either a low level of enzymatic activity on PGs suffices for repair of oxidative damage or the enzyme is more active on PG substrates in vivo than under our in vitro conditions. Our collaborative study with the LNT/NIMH, which is attempting to find yeast proteins that interact with Tdp1, is a potential avenue for identifying a putative activator. 3) Although hydrolysis of a 3'-blocking residue is a critical step, TDP1-dependent repair requires additional steps that are carried out by other proteins. To see if Tdp1 is the rate-limiting component in its repair pathway, we placed the gene downstream of an inducible promoter and examined the effect of overexpression on sensitivity to CPT and BLM. In no case did we find an improvement in survival; in fact, under some conditions overexpression of TDP1 decreased survival. This suggests that a later step in the repair pathway, such as removal of the 3'-phosphate generated by Tdp1 action, is rate-determining. Interestingly, overexpression of the inactive form of TDP1 can sensitize cells to DNA damage, sometimes dramatically so. We infer that, despite the alteration in its active site, the mutant protein can still bind to damaged residues. Since this binding must be sufficiently long-lived to block to other forms of repair, the mutant protein may be a useful tool for quantifying damage at the 3'-end.
| 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 |
