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. 2012 Apr 13;287(16):12848-57.
doi: 10.1074/jbc.M111.333963. Epub 2012 Feb 27.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells

Junko Murai  1 , Shar-yin N Huang, Benu Brata Das, Thomas S Dexheimer, Shunichi Takeda, Yves Pommier
Affiliations

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells

Junko Murai et al. J Biol Chem. .

Abstract

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.

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Figures

FIGURE 1.
FIGURE 1.
Alignment of human and chicken Tdp1 amino acid sequences. The sequences share 69% identity (asterisks) and 11% similarity (dots). Conserved catalytic residues are shown with bold letters and are indicated by arrowheads.
FIGURE 2.
FIGURE 2.
Targeted disruption of Tdp1 in DT40 cells and characterization of Tdp1−/− cells. A, schematic representation of the chicken Tdp1 locus and configuration of the targeted allele. Black squares indicate the exons. Two targeting plasmids, Tdp1-1-puro and Tdp1-2-hyg, with different homology arms and resistance genes were used. The restriction enzyme sites used for the Southern blot analysis are indicated. Primers (a–d) used for the RT-PCR in C are indicated by arrows. The probe used in the Southern blot analysis is indicated as a horizontal bar. loxP sites are indicated as black triangles flanking each resistance gene (white square). gg, gallus gallus. B, Southern blot analysis of NcoI- and NotI-digested genomic DNA from cells with the indicated genotypes using a flanking probe as shown in A. The arrowheads indicate the position of the detected band. C, representative RT-PCR analysis with the indicated genotypes using the paired primers shown in A. D, Western blot analysis of whole cell lysates prepared from the indicated genotypes. Blots were probed with anti-human TDP1 and anti-actin. E, growth curves of cells of the indicated genotypes. The error bars represent S.D. (n = 3). F, Tdp1 is the primary 3′-tyrosyl phosphodiesterase in DT40 cells. Top, scheme for the conversion of the 14-nt 3′-phosphotyrosyl substrate (14Y) to the 14-nt 3′-hydroxyl product (14OH) by sequential action of Tdp1 and polynucleotide kinase (PNKP). Bottom left, representative gel images of cleavage assays from the indicated cell lines. 5′-32P-Labeled 14Y (1 nm) was incubated with serially diluted (1:3) whole cell lysates. The highest concentration was 8.8 μg in a 10-μl reaction volume. Bottom right, quantification of processing activity for each cell lysate.
FIGURE 3.
FIGURE 3.
Involvement of Tdp1 for DNA repair induced by broad range of DNA-damaging agents. Survival curves of cells treated with the indicated DNA-targeting agents are shown. The indicated cells were treated with the indicated DNA-targeting agents continuously for 72 h before measuring ATP activity, which was used to measure cell viability. The survival of untreated cells was set as 100%. Error bars represent S.D. (n = 3). Gy, grays.
FIGURE 4.
FIGURE 4.
Processing activity of recombinant human TDP1 on double-stranded substrate harboring 5′-phosphotyrosyl linkage with blunt end (Y40/40) or 2- (Y40/38), 4- (Y40/36), or 6-base (Y40/34) 5′-overhangs. A, the substrates were incubated with 1 μm TDP1 for the indicated time. P40 was used as marker. A gel representative of consistent results in independent experiments is shown. B, quantification of TDP1 processing activity from A (left). The error bars represent the S.E. (n = 3). A scheme of the DNA substrates used in A is shown (right). An asterisk indicates the internal radiolabeled site.
FIGURE 5.
FIGURE 5.
Differential activity of recombinant human TDP1 for single-stranded DNA substrates harboring 5′-phosphotyrosine (Y19) and 5′-fluorescein (6-FAM19). A, scheme for the processing pathways of Y19 (left) and 6-FAM19 (right) by TDP1. Because of the nucleosidase activity of TDP1 (19), which removes the last 3′-end base, the Y19 substrate can be processed in two ways. One is that the 5′-tyrosine is removed first (P19) before the 3′-terminal adenine (P18) (clockwise). The other is that the 3′-adenine is removed first (Y18) before the 5′-tyrosine (P18) (counterclockwise). An asterisk indicates the radiolabeled site. B, each substrate was incubated with serial dilutions (1:3) of TDP1 at 25 °C for 30 min with the highest TDP1 concentration starting at 1 μm. Processing of 6-FAM19 by TDP1 yielded one product (right), whereas processing of Y19 yielded two distinct products (left), indicating that TDP1 processes 5′-phosphotyrosine but not 5′-fluorescein.
FIGURE 6.
FIGURE 6.
Involvement of Tdp1 for repair of abasic (AP) sites. A, scheme for the conversion of MMS- and H2O2-induced DNA damage into the substrates for Tdp1. DNA adducts like methylated base and 8-oxoguanine (8-oxo) are converted into AP sites by DNA glycosylase. In one way, Tdp1 cleaves the AP site, directly generating the 3′-phosphate end. In the other way, the AP site turns out to be a single strand break with a 3′-dRP end after hydrolysis by AP lyase. Tdp1 cleaves the 3′-dRP, generating the 3′-phosphate end. B, processing activity of recombinant human TDP1 on the 14-nt 3′-phosphotyrosyl substrate (14Y) and the 14-nt 3′-dRP substrate (14dRP). Each substrate was incubated with serial dilutions (1:3) of TDP1 at 25 °C for 30 min with the highest concentration starting at 30 nm. The 14-nt 3′-phosphate (14P) was used as a marker. A gel representative of consistent results in independent experiments is shown. C, quantification of TDP1 processing activity for each substrate from B. EC50 values (the half-maximal effective concentration) for 14Y and 14dRP are 0.062 and 1.078 nm, respectively.
FIGURE 7.
FIGURE 7.
Interactions between CtIP and Tdp1 for repair of Top1cc, Top2cc, and MMS-induced damage. A–C, survival curves of cells treated with CPT (A), etoposide (B), or MMS (C). Sensitivity was determined as in Fig. 3. The survival of untreated cells was set as 100%. Error bars represent S.D. (n = 3).
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