Deficiency in 3'-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)

Abstract

Tyrosyl-DNA phosphodiesterase (TDP1) is a DNA repair enzyme that removes peptide fragments linked through tyrosine to the 3' end of DNA, and can also remove 3'-phosphoglycolates (PGs) formed by free radical-mediated DNA cleavage. To assess whether TDP1 is primarily responsible for PG removal during in vitro end joining of DNA double-strand breaks (DSBs), whole-cell extracts were prepared from lymphoblastoid cells derived either from spinocerebellar ataxia with axonal neuropathy (SCAN1) patients, who have an inactivating mutation in the active site of TDP1, or from closely matched normal controls. Whereas extracts from normal cells catalyzed conversion of 3'-PG termini, both on single-strand oligomers and on 3' overhangs of DSBs, to 3'-phosphate termini, extracts of SCAN1 cells did not process either substrate. Addition of recombinant TDP1 to SCAN1 extracts restored 3'-PG removal, allowing subsequent gap filling on the aligned DSB ends. Two of three SCAN1 lines examined were slightly more radiosensitive than normal cells, but only for fractionated radiation in plateau phase. The results suggest that the TDP1 mutation in SCAN1 abolishes the 3'-PG processing activity of the enzyme, and that there are no other enzymes in cell extracts capable of processing protruding 3'-PG termini. However, the lack of severe radiosensitivity suggests that there must be alternative, TDP1-independent pathways for repair of 3'-PG DSBs.

Figure 1

Substrates with 3′-PG termini. (A) Oligomeric substrate. The 3′-PG terminus can be converted to a 3′-phosphate by TDP1 and thence to a 3′-OH by PNKP. (B) The 5.5 kb 3′-PG plasmid substrate is internally labeled, and 3′-terminal processing can be monitored by cleaving the substrate with AvaI and analyzing the labeled fragment by denaturing gel electrophoresis.

Figure 2

Processing of a 3′-PG 14mer by extracts of lymphoblastoid cells from SCAN1 patients (closed symbols) and from unaffected members (open symbols) of the same family, including TDP1+/– carriers (squares, males; circles, females). The substrate was treated with 0.3, 1 or 3 mg/ml of each whole-cell extract (A), or with 0.14, 1.4 or 5 mg/ml of each nuclear extract (B) for 2 h in the presence of EDTA. Following heat denaturation of cellular proteins, the substrate was treated with PNKP to convert 3′-phosphate to 3′-OH termini. Titration with various concentrations of extract (C) showed that whole-cell extracts (closed circles) contained about twice as much PG-processing activity as nuclear extracts (open squares).

Figure 3

Processing of a plasmid bearing partially complementary PG-terminated 3-base 3′ overhangs by normal and SCAN1 extracts. The substrate was treated for the indicated times with 3 mg/ml whole-cell (A) or 1.8 mg/ml nuclear extract (B), deproteinized, treated (or not) with PNKP, cut with AvaI and subjected to denaturing gel electrophoresis. Some samples also contained 1 mM dimethylaminopurine (DMAP), 10 μM wortmannin (Wort) and/or 2% dimethyl sulfoxide (DMSO, solvent for wortmannin). The lanes marked (asterisk) in (B) indicate samples that were treated in the reaction buffer normally used for whole-cell extract. In (B), all samples treated with the normal extract showed 1.8–2.1% conversion to 3′-OH, while those treated with the SCAN1 extract showed no detectable conversion. A trace of 14-PG fragment seen in some samples is due to residual single-strand oligomer that was not incorporated into the plasmid.

Figure 4

Reconstitution of 3′-PG processing and subsequent gap filling in SCAN1 extracts by recombinant TDP1. The plasmid substrate was treated with 3 mg/ml SCAN1 whole-cell extract (subject 1635) and analyzed as in Figure 3. In some cases, 25 ng of recombinant TDP1 and/or 200 ng of XRCC4/DNA ligase IV complex (Trevigen) were added. Some samples contained 5 mM EDTA instead of 0.5 mM MgCl₂, as indicated.

Figure 5

Phosphorylation of TDP1. Cells irradiated with 0 or 20 Gy were incubated in the presence of [³²P]orthophosphate for the indicated times. Affinity-purified FLAG-TDP1 was subjected to denaturing gel electrophoresis followed by phosphorimaging (A) or western blotting with FLAG antibody (B).

Figure 6

Dephosphorylation of TDP1 by PP1. Affinity-purified phosphate-labeled TDP1 from cytoplasmic or nuclear extracts of cells treated with 0 or 20 Gy ionizing radiation was exposed to PP1 and/or YOP phosphatase.

Figure 7

Radiosensitivity of SCAN1 cells. Plateau-phase normal cells (closed circles, subject 1646) or SCAN1 cells (open squares, subject 1664) were irradiated daily for 5 days with 0 (A), 0.4 (B), 0.8 (C) or 1.6 (D) Gy of γ-rays, then diluted to 10⁵/ml for assessment of cell growth. (E–H) Identical experiment with another normal and another SCAN1 cell line (subjects 1668 and 1662, respectively).