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. 2001 Oct 9;98(21):12009-14.
doi: 10.1073/pnas.211429198. Epub 2001 Sep 25.

The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily

H Interthal  1 , J J Pouliot, J J Champoux
Affiliations

The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily

H Interthal et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Sequence alignment of Tdp1 orthologs. The program pileup of the GCG package was used with some manual modification to create the alignment. boxshade was used to shade residues black that are identical in ≥ 50% of the sequences and similar residues were shaded in gray. The HKD motifs are indicated by black bars above the alignment. Abbreviations and accession numbers are as follows: hs (Homo sapiens Tdp1 predicted protein FLJ11090, National Center for Biotechnology Information RefSeq accession no. NP_060789), ce (C. elegans, GenPept: AAC68960), dm (D. melanogaster glaikit protein, GenPept: CAB86488), at (A. thaliana, GenPept: CAB89327), sp (S. pombe, hypothetical protein SPCP31B10.05, PIR: T41695), sc (S. cerevisiae Tdp1, gene product of ORF YBR223c, GenPept: CAA85186). The C. elegans and A. thaliana protein sequences were predicted based on genomic sequences.
Figure 2
Figure 2
Alignment of the HKD motifs found in Tdp1 orthologs with the HKD motifs of a representative member from each PLD superfamily class. The program clustalw with additional manual modifications was used to create the alignment. Proteins that possess only one HKD motif are grouped with the N-terminal motifs of the proteins containing two HKD motifs. Highlighted in gray are key residues that fall into one of the three categories established by Ponting and Kerr (1) for the members of the PLD superfamily: hydrophobic residues (A, C, I, L, V, M, F, Y, W), small residues (G, A, S), and acidic and amides of acidic residues (D, E, N, Q). Black boxes indicate the most conserved amino acids in the motif. * marks positions that were mutated in human Tdp1. Numbers flanking the sequences indicate the positions of the first and last amino acids shown in that protein sequence. Numbers in brackets represent the numbers of amino acids not shown in the alignment. Accession codes and sequences represented are the same as in Fig. 1 for the Tdp1 orthologs. Representatives of the other PLD superfamily classes are [roman numerals indicate the class according to Ponting and Kerr (1)]: PLD_cb (I, castor bean PLD, PIR: A54850), Toxin_yp (II, Y. pestis murine toxin, GenPept: CAA63386), CLS_ec (III, E. coli cardiolipin synthase, SWISS-PROT: P31071), PSS_ec (IV, E. coli phosphatidylserine synthase, SWISS-PROT: P23830), orf_ssp (VI, Synchocystis sp. product of comE ORF1, GenPept: BAA10416), nuc_st (VII, S. typhimurium Nuc protein precursor, PIR: S41475), o338_ec (VIII, E. coli hypothetical 38-kDa protein in FEC1-FIMB intergenic region, SWISS-PROT: P39369), p37K_vv (V, Vaccinia virus p37K major envelope protein, SWISS-PROT: P20638).
Figure 3
Figure 3
Tdp1 activity assay. (A) Schematic representation of substrate preparation and Tdp1 activity assay. The 5′ end-labeled topoisomerase I oligonucleotide suicide substrate CL14N/CP25N is cleaved by topoisomerase I (site indicated with arrow), and the 12-mer cleavage product remains covalently bound to the topoisomerase (12-topo). Subsequent digestion with trypsin leaves a small peptide covalently bound to the DNA via the active site tyrosine. This is the substrate (12-pep) for Tdp1, which cleaves specifically between the peptide and the DNA to produce a 12-mer oligonucleotide with a 3′ phosphate group (12-P). * indicates the 32P 5′ end-labeled scissile strand (CL14N). (B) Sequencing gel analysis of reaction products. Serial 5-fold dilutions of the various proteins (as indicated by the triangles above the lanes) were incubated for 15 min with identical amounts of substrate 12-pep (lane 1). The band labeled CL14N present in every lane is unreacted 14-mer suicide substrate. The product of the Tdp1 hydrolysis is 12-P. The identities of the bands were verified by using oligonucleotides with known structures (data not shown).
Figure 4
Figure 4
Identification of a covalent intermediate in the Tdp1 reaction. (A) SDS/PAGE analysis of reactions stopped at the indicated times with SDS. The covalent reaction intermediate is labeled 12-Tdp1. The faint band labeled 12-topo consists of residual undigested topo I covalently bound to the 12-mer (12-topo, Fig. 3A). (B) Sequencing gel analysis of the same time-course experiment samples as in A. Bands are labeled as in Fig. 3B. (C) SDS/PAGE analysis of reaction intermediates trapped with SDS for wild-type (wt) and Δ1–148 Tdp1. Both proteins are shifted to a slower mobility when they are covalently bound to the substrate.
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