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. 2005 Oct 28;280(43):36518-28.
doi: 10.1074/jbc.M508898200. Epub 2005 Aug 31.

Human Tdp1 cleaves a broad spectrum of substrates, including phosphoamide linkages

Affiliations

Human Tdp1 cleaves a broad spectrum of substrates, including phosphoamide linkages

Heidrun Interthal et al. J Biol Chem. .

Abstract

Human tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety. In eukaryotic cells, this type of linkage is found in stalled topoisomerase I-DNA covalent complexes, and Tdp1 has been implicated in the repair of such complexes in vivo. We confirm here that the Tdp1 catalytic cycle involves a covalent reaction intermediate in which a histidine residue is connected to a DNA 3'-phosphate through a phosphoamide linkage. Most surprisingly, this linkage can be hydrolyzed by Tdp1, and unlike a topoisomerase I-DNA complex, which requires modification to be an efficient substrate for Tdp1, the native form of Tdp1 can be removed from the DNA. The spinocerebellar ataxia with axonal neuropathy neurodegenerative disease is caused by the H493R mutant form of Tdp1, which shows reduced enzymatic activity and accumulates the Tdp1-DNA covalent intermediate. The ability of wild type Tdp1 to remove the stalled mutant protein from the DNA likely explains the recessive nature of spinocerebellar ataxia with axonal neuropathy. In addition to its activity on phosphotyrosine and phosphohistidine substrates, Tdp1 also possesses a limited DNA and RNA 3'-exonuclease activity in which a single nucleoside is removed from the 3'-hydroxyl end of the substrate. Furthermore, Tdp1 also removes a 3' abasic site and an artificial 3'-biotin adduct from the DNA. In combination with earlier data showing that Tdp1 can use 3'-phosphoglycolate as a substrate, these data suggest that Tdp1 may function to remove a variety of 3' adducts from DNA during DNA repair.

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Figures

Fig. 1
Fig. 1
Chemical characterization of the phosphoamino acid bond in the Tdp1-DNA covalent intermediate. A. Schematic representation of substrate preparation. B. SDS-PAGE of Tdp1 H493R-DNA covalent reaction intermediate (12-Tdp1) samples treated under the indicated conditions. C. SDS-PAGE of treated topoisomerase I-DNA covalent complexes (12-Topo).
Fig. 2
Fig. 2
Tdp1 activity assay on the phosphoamide bond between a DNA 3′ phosphate and a Tdp1 tryptic peptide. A. 20-TDPpep (lane 2) was generated by incubating the H493R mutant protein with 20-TOPOpep (lane 1) followed by trypsin digestion. 20-TDPpep was incubated with wt Tdp1 (lane 3) or Tdp1 H263A (lane 4) followed by sequencing gel analysis. B. Structure of the DNA-topoisomerase I tryptic peptide complex in which the topoisomerase I fragment (TOPOpep) is linked to the DNA 3′ phosphate through a phosphodiester bond with the active site tyrosine. C. Structure of the DNA-Tdp1 tryptic peptide complex. The Tdp1 peptide (TDPpep) is connected to the DNA via a phosphoamide bond between the nucleophilic His263 and the 3′ DNA phosphate.
Fig. 3
Fig. 3
Tdp1 activity on topoisomerase I-DNA and Tdp1-DNA covalent complexes. A. Tdp1 activity assays on topo70-DNA covalent complexes that were either untreated (lanes 3-6), incubated at 65°C for 10 min (lanes 7-10), or SDS denatured (lanes 11-13) before Tdp1 addition. At the indicated times samples were taken, the reactions stopped with SDS, and trypsin digested before sequencing gel analysis. In lane 1, 20-TOPOpep was loaded as a size marker for the 20-mer DNA with a tryptic topoisomerase I peptide. Lane 2 contains the product of the reaction of wt Tdp1 on 20-TOPOpep, a 20-mer with a 3′ phosphate group (20-P). B. Tdp1 activity assay on SDS denatured Tdp1-DNA complexes. 20-TOPOpep was incubated with Tdp1 H493R and the reaction stopped with SDS (lane 1). To visualize the covalent Tdp1-DNA intermediate, a portion of the reaction was digested with trypsin before loading (lane 4). Wt (lane 2) or H263A (lane 3) Tdp1 were added to the substrate mix shown in lane 1 and the reactions were analyzed on a sequencing gel.
Fig. 4
Fig. 4
Activities of wt and Tdp1 mutants on the H493R-DNA reaction intermediate. A. Tdp1 H493R and 20-TOPOpep were incubated for the indicated times, treated with trypsin and analyzed on a sequencing gel. B. H493R was incubated with 20-TOPOpep (lane 1) for 2 min (lane 2) which serves as the zero time point for the subsequent reactions. At this time point , the indicated second enzymes (lanes 8-27) were added to the reaction mixtures and samples were taken at the time points shown. No second enzyme was added to the time course experiment shown in lanes 3-7. All samples were digested with trypsin, and analyzed on a sequencing gel. C. The accumulation of the reaction product 20-P over time is plotted for the reactions shown in B. H493R alone (filled squares), plus wt (open circles), plus additional H493R (open squares), plus Δ(1-148) wt (filled circles), and plus H263A (open triangles).
Fig. 5
Fig. 5
The DNA in the covalent reaction intermediate is “handed-off” from one Tdp1 molecule to the other. A. Either full length H493R or N-terminally truncated Δ(1-148) H493R was incubated with 20-TOPOpep for 2 min to form 20-Tdp FL and 20-Tdp Δ, respectively. The indicated second enzyme was added to the reactions at 2 min and referred to as the zero time point for the subsequent time course analyses. Samples were taken at the time points shown and analyzed by SDS-PAGE. B. The decay of the Tdp1-DNA intermediate over time as determined from a parallel sequencing gel analysis is plotted for a subset of the time course experiments shown in (A). Averages of three experiments are shown with error bars representing standard deviations. Kinetics of decay are shown for reactions containing additional H493R (open squares), and after the addition of wt Tdp1 (open circles)). The decay of the double-stranded DNA-Tdp1 H493R covalent complex in the absence of excess free Tdp1 H493R was plotted over time (filled triangles).
Fig. 6
Fig. 6
Tdp1 activity assays on DNA and RNA substrates. A. A 20-mer DNA oligonucleotide with a 3′ hydroxyl (lane 1) was incubated with either wt (lane 4) or H263A (lane 5) Tdp1. A 19-mer with a 3′ phosphate was 5′ end-labeled with either wt PNK (lane 2) or a 3′ phosphatase minus mutant PNK (PNK, lane 3) to obtain size markers for a 19-mer with a 3′ hydroxyl (19-OH) and a 19-mer with a 3′ phosphate group (19-P), respectively. For the samples loaded in lanes 6-8, the 20-mer DNA was first incubated with the indicated Tdp1 variant and then treated with PNK (lanes 7 and 8) or left untreated (lane 6). The samples were analyzed on a sequencing gel. B. A 29-mer RNA oligonucleotide annealed to a 48-mer DNA nucleotide (lane 1) was incubated with either wt Tdp1 (lane 4) or Tdp1 H263A (lane 5) before sequencing gel analysis. The RNA ladders with either 3′ hydroxyl or 3′ phosphate groups were generated by incubating the 29-mer RNA with P1 nuclease (lane 2) or NaOH (lane 3), respectively.
Fig. 7
Fig. 7
Tdp1 activity assays on DNA oligonucleotides with 3′ tetrahydrofuran or 3′ biotin moieties. A. Structure of the abasic site mimic tetrahydrofuran on the DNA 3′ end. B. A 20-mer oligonucleotide containing the tetrahydrofuran moiety on the 3′ end (lane 3, 20-AB) was incubated with wt Tdp1 (lane 4) or the H263A mutant enzyme (lane 5). A corresponding 20-mer with a 3′ hydroxyl group (lane 1, 20-OH) was used as a size marker. The 20-mer with a 3′ phosphate (20-P) was generated by incubating a 20-mer with a 3′ tyrosyl moiety with wt Tdp1 (lane 2). C. Structure of biotin connected to the 3′ end of the DNA by a 15-atom linker. D. Wt Tdp1 (lane 4) or H263A (lane 5) were incubated with a 3′ biotinylated 20-mer DNA (lane 1, 20-biotin) and analyzed on a sequencing gel. To generate size markers (20-OH and 20-P),` a corresponding 3′ phosphorylated 20-mer was labeled with either wt PNK (lane 2) or a 3′ phosphatase minus mutant PNK (PNK) (lane 3).

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References

    1. Yang SW, Burgin AB, Jr., Huizenga BN, Robertson CA, Yao KC, Nash HA. Proc. Natl. Acad. Sci. U.S.A. 1996;93:11534–11539. - PMC - PubMed
    1. Wang JC. Annu. Rev. Biochem. 1996;65:635–692. - PubMed
    1. Wang JC. Nat Rev Mol Cell Biol. 2002;3:430–440. - PubMed
    1. Champoux JJ. Annu. Rev. Biochem. 2001;70:369–413. - PubMed
    1. Nitiss JL. Biochim. Biophys. Acta. 1998;1400:63–81. - PubMed

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