The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.T he members of the phospholipase D (PLD) superfamily comprise a highly diverse group of proteins that include plant, mammalian and bacterial PLDs, bacterial phosphatidylserine and cardiolipin synthases, a bacterial toxin, several poxvirus envelope proteins, and some bacterial nucleases (1-3). Sequence alignments reveal that with the exception of two nucleases (1) the proteins arose as a result of a gene duplication event with each half of the protein containing four repeated motifs. Motifs 3 and 4 contain the highly conser ved HxK(x) 4 D(x) 6 GSxN sequence, termed the HKD motif (4), which has been implicated in the catalytic mechanism (see below). The crystal structure of the Salmonella typhimurium Nuc protein (4), one of the bacterial nucleases, shows that the active form of the enzyme is a dimer with the HKD motifs contributed by each subunit organized in roughly the same spatial arrangement as the two HKD motifs found in the crystal structure of the monomeric PLD from Streptomyces sp. strain PMF (5).For those members of the superfamily known to have catalytic activity, the enzymatic reactions all involve phosphoryl transfer from a donor to an acceptor that is either an alcohol or water. In the cases of the phospholipid synthases, a phosphatidyl moiety is transferred either from a cytidine 5Ј-diphosphate-diacylglycerol donor to serine or from phosphatidylglycerol to another phosphatidylglycerol to synthesize phosphatidylserine or cardiolipin, respectively. The PLDs either hydrolyze the phosphodiester bond in the phospholipid to produce phosphatidic acid and a free head group (often choline) or catalyze the exchange of one head group for another (transphosphatidylation). The nucleases appear to simply catalyze the hydrolysis of DNA phosphodiester bonds.The similar chemistry underlying these reactions suggests that the enzymes in the superfamily share a similar catalytic mechanism. Early evidence showing retention of configuration at the substrate phosphorous in the re...

Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death. Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described. Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair. The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized. The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.

Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3 end of broken DNA. The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist. Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage. In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase. However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair. We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered.

Mammalian cells contain potent activity for removal of 3-phosphoglycolates from single-stranded oligomers and from 3 overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a singlestranded 3 terminus to leave a 3-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3-phosphoglycolate on a 3-base 3 overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3 overhangs.Ionizing radiation, radiomimetic drugs, and to some extent all free radical-based genotoxins induce DNA double strand breaks (DSBs) 1 by oxidative fragmentation of DNA sugars (1-4). Most such breaks bear terminal 3Ј-phosphate or 3Ј-phosphoglycolate (PG) moieties (1, 2, 5) that must be removed to allow fill-in of gaps by DNA polymerase and final religation by DNA ligase. While the human apurinic/apyrimidinic endonuclease Ape1 can remove PGs, albeit inefficiently, from blunt and recessed 3Ј ends of DSBs (6), PGs on 3Ј overhangs are highly resistant to this enzyme (6, 7). Nevertheless, in mammalian cell extracts, PGs on 3Ј overhangs of DSBs are removed by an as yet unidentified activity, and a fraction of such DSBs are accurately rejoined by Ku-mediated end alignment, gap-filling, and ligation (8).The yeast tyrosyl-DNA phosphodiesterase scTdp1 was isolated from Saccharomyces cerevisiae as an activity that hydrolyzes the phosphodiester bond linking tyrosine to a 3Ј DNA end (9). Such linkages are formed as intermediates in DNA relaxation by topoisomerase I and in DNA cleavage and reunion by various recombinases. These intermediates can become trapped if the process is interrupted, for example, by a topoisomerase inhibitor or by collision with a replication fork. The hypersensitivity of tdp1⌬ yeast to topoisomerase I-mediated DNA damage (10) suggests a critical role for scTdp1 in repair of such lesions. A human homologue with similar 3Ј-phosphotyrosyl-processing activity, hTdp1, was cloned by homology to scTdp1 and was eventually shown to have some homology to the phospholipase D superfamily of phosphodiesterases (11). Yeast and human Tdp1 differ from other activities for processing blocked 3Ј ends in that they leave a 3Ј-phosphate rather than a 3Ј-hydroxyl. A 3Ј-phosphate could then be removed by polynucleotide kina...