Normal rates of chromosomal SSBR in cells lacking aprataxin. (A) AOA1 lymphoblastoid cells were incubated in the absence (−) or presence (+) of 50 μM H₂O₂ (15 min on ice), followed by incubation in a drug-free medium for the indicated repair period at 37°C. Chromosomal DNA strand breaks were then measured by alkaline comet assays. (B) AOA1 primary fibroblasts were treated in the absence (−) or presence (+) of 75 μM H₂O₂ (15 min on ice) and processed as described for panel A. Insets in panels A and B contain the same data as in the main panels but plotted as percent damage remaining. (C) WT, Aptx^−/−, and Xrcc1^−/− primary quiescent astrocytes were incubated in the absence (−) or presence (+) of 75 μM H₂O₂ (10 min on ice), followed by incubation in a drug-free medium for the indicated repair period at 37°C. Chromosomal DNA strand breaks were then measured by alkaline comet assays. (D) WT, Aptx^−/−, and Xrcc1^−/− primary quiescent astrocytes were incubated in the absence (−) or presence of the indicated concentration of MMS (10 min at 37°C). Chromosomal DNA strand breaks were then measured by alkaline comet assays.

Removal of 5′ AMP by aprataxin is not required for end processing by PNK or gap filling by Pol β. (A) Cartoon of aprataxin-dependent short-patch repair of adenylated SSBs in WT cells. (B) 5′ AMP does not affect processing of 3′-phosphate termini by PNK. An SSB substrate (25 nM) lacking or containing 5′ AMP as indicated (top) was incubated either with 25 nM, 50 nM, 125 nM, or 250 nM recombinant human PNK or without PNK (−) for 1 h at 30°C. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. Note that the 5′ terminus of the 17-mer is labeled with ³²P in these experiments to allow detection of 3′ end processing. The position of the 17-mer harboring 3′ phosphate (17-P) or 3′ hydroxyl (3′-OH) is indicated. (C) 5′ AMP does not affect gap filling by Pol β. An SSB substrate (25 nM) lacking or containing 5′ AMP as indicated (top) was incubated either with 5 nM, 15 nM, 30 nM, or 100 nM purified recombinant human Pol β or without Pol β (−) for 1 h at 30°C. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. The positions of the 17-mer and 18-mer are indicated as markers. (D) Cartoon depicting APTX-independent repair of adenylated SSBs in AOA1. Note the hypothetical blockage of this process at the final step of DNA ligation.

Complementation of the SSBR defect in AOA1 lymphoblastoid cell extracts by recombinant human Lig3α and reconstitution of aprataxin-independent SSBR with recombinant human proteins. (A) Complementation of the SSBR defect in AOA1 lymphoblastoid cell extracts by recombinant DNA ligase IIIα (Lig3α). Total WT or AOA1 lymphoblastoid cell extracts (5 μg) were incubated with a ³²P-labeled 5′-AMP SSB (100 nM) substrate for 1 h at 30°C in the presence or absence of 1 mM ATP and/or 8 nM, 24 nM, or 72 nM Lig3α, as indicated. (B) Reconstitution of aprataxin-independent SSBR with recombinant human proteins. A 5′-AMP SSB substrate (60 nM) was incubated with 250 nM recombinant PNK, 100 nM Pol β, and 80 nM Lig3α in the presence or absence of 100 nM aprataxin (APTX) and 1 mM ATP, as indicated, for 1 h at 30°C. Reaction products were fractionated by denaturing PAGE and detected by phosphorimager.

Aprataxin-dependent short-patch SSBR of 5′-AMP SSBs in AOA1 lymphoblastoid extracts. (A) Defective short-patch SSBR in AOA1 extracts and its complementation by recombinant aprataxin. (Top) Cartoon of the oligonucleotide duplex employed for these experiments, containing a 1-bp gap, a 3′-phosphate terminus, and a 5′-AMP terminus. The 5′ phosphate to which AMP is covalently linked is labeled with ³²P. The position of this label restricts the assay to measurements of short-patch repair events only. Nucleotide lengths are shown. (Bottom) Portions (0.1 μg, 1.0 μg, or 5.0 μg) of either WT (ConR2) or AOA1 (Ap5) cell extracts were incubated for 60 min at 30°C with a ³²P-labeled 5′-AMP SSB substrate (25 nM) in the absence (left) or presence (right) of recombinant human aprataxin (APTX) (100 nM). Reaction products were fractionated and detected as described in Materials and Methods. The ³²P-labeled 25-mer containing (AMP-³²P-25) or lacking (³²P-25) 5′ AMP was fractionated in parallel for size markers (Mks). The position of the repaired ³²P-labeled 43-mer is also indicated. Asterisks indicate nonspecific nucleolytic products. (B) Normal short-patch repair of SSBs lacking 5′ AMP in AOA1 cell extracts. (Top) Cartoon of the oligonucleotide duplex employed for these experiments, containing a 1-bp gap, a 3′-phosphate terminus, and a normal ³²P-labeled 5′-phosphate terminus. WT (ConR2) or AOA1 (Ap5) cell extracts (5 μg) were incubated for 60 min at 30°C with a ³²P-labeled SSB substrate lacking 5′ AMP (100 nM). Reaction products were fractionated and detected as described in Materials and Methods.

Short-patch SSBR fails in AOA1 due to insufficient levels of nonadenylated DNA ligase. (A) AOA1 cell extracts cannot efficiently ligate adenylated DNA nicks. Total WT (ConR2) or AOA1 (Ap5) lymphoblastoid cell extracts (6.25 μg) were incubated with a ³²P-labeled adenylated nicked substrate (25 nM) (top) for the indicated times at 30°C. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. A ³²P-labeled 25-mer containing 5′ AMP (AMP-25) and one lacking 5′ AMP were fractionated for size markers (Mks). The position of the 43-mer ligated product is indicated. (B) Cartoon depicting ligation of a nonadenylated DNA nick by DNA ligase. (i) An adenylated DNA ligase molecule transfers AMP to the 5′-phosphate terminus of a DNA nick, resulting in an adenylated nick and a nonadenylated ligase molecule. (ii) The nonadenylated DNA ligase molecule rapidly catalyzes a nucleophilic attack of the pyrophosphate bond linking AMP to DNA, resulting in nick ligation and the release of AMP. The nonadenylated DNA ligase molecule is readenylated (dashed arrow) in readiness for the next ligation event. (C) Complementation of the SSBR defect in AOA1 lymphoblastoid extracts by recombinant T4 DNA ligase. Total WT (ConR2) or AOA1 (Ap5) lymphoblastoid cell extracts (5 μg) were incubated with a ³²P-labeled 5′-AMP SSB substrate (25 nM) for 1 h at 30°C in the presence or absence of 1 mM ATP and/or 2 U of T4 DNA ligase, as indicated. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. The positions of the ³²P-labeled 43-mer reaction product and the ³²P-labeled 25-mer containing or lacking 5′ AMP are indicated. M'rkers, markers. (D) Complementation of the SSBR defect in Aptx^−/− primary astrocyte extracts by recombinant T4 DNA ligase. Aptx^+/+ (+/+) or Aptx^−/− (−/−) mouse astrocyte extracts (5 μg) were incubated with a ³²P-labeled 5′-AMP SSB substrate (25 nM) for 1 h at 30°C in the presence or absence of 1 mM ATP and/or 2 U of T4 DNA ligase, as indicated. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. The positions of the ³²P-labeled 43-mer reaction product and the ³²P-labeled 25-mer containing or lacking 5′ AMP are indicated.

Defective short-patch SSBR in quiescent primary Aptx^−/− mouse neural cells. (A) Quiescent WT and Aptx^−/− mouse astrocytes were either left untreated (−) or treated with 75 μM H₂O₂ for 10 min on ice (+) and, where indicated, then incubated in drug-free medium for 30 or 60 min. Where indicated (+Aph), cells were preincubated with 50 μM aphidicolin for 30 min at 37°C, and repair was conducted in the continued presence of aphidicolin. Chromosomal DNA strand breaks were then quantified by alkaline comet assays. Each bar represents the average mean tail moment from three independent experiments (error bars, ±1 standard deviation). Asterisks indicate statistically significant differences (*, P ≤ 0.05; **, P ≤ 0.01). (B) Quiescent WT and Aptx^−/− mouse astrocytes were either left untreated (−) or treated with 25, 50, or 100 μM MMS for 10 min at 37°C with (+Aph) or without preincubation with 50 μM aphidicolin. Chromosomal DNA strand breaks were then quantified by alkaline comet assays. Each bar represents the average mean tail moment from three independent experiments (error bars, ±1 standard deviation). Asterisks indicate statistically significant differences (*, P ≤ 0.05; **, P ≤ 0.01).