Essential role for mammalian apurinic/apyrimidinic (AP) endonuclease Ape1/Ref-1 in telomere maintenance

Abstract

The major mammalian apurinic/apyrimidinic endonuclease Ape1 is a multifunctional protein operating in protection of cells from oxidative stress via its DNA repair, redox, and transcription regulatory activities. The importance of Ape1 has been marked by previous work demonstrating its requirement for viability in mammalian cells. However, beyond a requirement for Ape1-dependent DNA repair activity, deeper molecular mechanisms of the fundamental role of Ape1 in cell survival have not been defined. Here, we report that Ape1 is an essential factor stabilizing telomeric DNA, and its deficiency is associated with telomere dysfunction and segregation defects in immortalized cells maintaining telomeres by either the alternative lengthening of telomeres pathway (U2OS) or telomerase expression (BJ-hTERT), or in normal human fibroblasts (IMR90). Through the expression of Ape1 derivatives with site-specific changes, we found that the DNA repair and N-terminal acetylation domains are required for the Ape1 function at telomeres. Ape1 associates with telomere proteins in U2OS cells, and Ape1 depletion causes dissociation of TRF2 protein from telomeres. Consistent with this effect, we also observed that Ape1 depletion caused telomere shortening in both BJ-hTERT and in HeLa cells. Thus, our study describes a unique and unpredicted role for Ape1 in telomere protection, providing a direct link between base excision DNA repair activities and telomere metabolism.

Keywords: chromosome stability; endogenous DNA damage; genome stability.

Fig. 1.

Down-regulation of Ape1 is associated with mitotic defects in U2OS cells. (A) Depletion of Ape1 induces mitotic defects. U2OS cells treated with the indicated siRNAs were fixed at 72 h posttransfection and immunostained for Ape1 and α-tubulin. DNA was stained with DAPI. (Scale bar, 20 µm.) (B) Percentages of U2OS cells with multilobular nuclei, micronuclei, and bi/multinuclei were quantified at 72 h of the siRNA treatments. Error bars represent the SDs of three independent transfections. *P ≤ 0.05; **P ≤ 0.01 (Student t test). (C) Endogenous (Ape1) and GFP fusion variants of Ape1 (GFP-Ape1) were detected by immunoblotting in the virally transduced U2OS cells following five days of drug selection. (D) RNAi-resistant Ape1 and the C65S mutant Ape1, but not the K6A/K7A and the N212A mutant Ape1, partially rescued the mitotic defects of Ape1-depleted cells. U2OS cells expressing GFP-fusion variants of Ape1 were transfected with either control siRNA or APE1 siRNA-1 at day 5 of drug selection. Multilobular nuclei, micronuclei, bi/multinucleated cells, and giant cell nuclei were quantified at 72 h of the siRNA transfection. At least 100 cells were counted for each experiment, and the SDs of three independent experiments were calculated. Statistical significance between the cells expressing wild-type Ape1 and the cells expressing mutant forms of Ape1 is shown. *P ≤ 0.05 (Student t test).

Fig. 2.

Ape1 depletion causes chromosome segregation defects and telomere dysfunction. (A) Chromosome segregation defects in Ape1-depleted cells. U2OS cells were transfected with control (Ctr.) or APE1 siRNAs and synchronized by double thymidine treatment. At day 3, the cells were released into fresh medium and fixed after 9 h. DNA was stained with PI. (Scale bar, 10 µm.) (B) The percentage of cells representing three examples of typical chromosome segregation defects is shown. Error bars denote the SDs of three independent experiments. *P ≤ 0.05 (Student t test). (C) Telomere aberrations in Ape1-depleted cells. U2OS cells treated with control or APE1 siRNAs were synchronized by the double thymidine treatment, and released into nocodazole (100 ng/mL) for 16 h (Fig. S4). Telomeres were labeled with a PNA probe (red). Representative images of telomere aberrations observed in Ape1-depleted cells are shown. At least 50 metaphases per experiment were counted, and SDs were calculated from three independent transfections. *P ≤ 0.01 (Student t test). (D) The DNA repair and gene regulatory domains of Ape1 are required for telomere protection. U2OS cells were infected with the viruses expressing the wild-type (WT) or the mutant forms of Ape1: K6A/K7A, C65S, and N212A. Following a 7 d-drug selection, metaphase chromosomes were prepared, and telomeres were labeled with a PNA telomere C-probe (green). DNA was stained with DAPI. (Scale bar, 5 µm.) From three independent infections, at least 50 metaphases per experiment were analyzed, and metaphases showing three or more end-to-end chromosome fusions were quantified. *P ≤ 0.01 (Student t test).

Fig. 3.

Ape1 is required for stabilization of telomeric DNA. (A) Ape1 depletion causes TRF2 loss at telomeres. Control (Ctr.) and APE1 siRNA transfected U2OS cells were fixed at 72 h posttransfection and immunostained for Ape1 (green) and TRF2 (red). DNA was stained with DAPI. (Scale bar, 10 µm.) (B) Telomere-ChIP assay was performed in U2OS cells at 72 h of siRNA transfection. The recovered DNA from chromatin immunoprecipitation with Ape1 or TRF2 antibodies was submitted to quantitative real-time PCR analysis. Telomeric DNA was amplified using a telomere-specific primer pair, and the results are presented as the percentage of input DNA. Error bars correspond to SDs from three independent experiments. (C) Ape1 depletion induces DNA damage response at telomeres. BJ-hTERT cells transfected with control (Ctr.) or APE1 siRNAs were fixed at 72 h posttransfection and immunostained for TRF1 (green) or γH2AX (red). DNA was stained with DAPI. (Scale bar, 5 µm.) Cells showing more than three TRF2/γH2AX colocalizing foci from three independent, blinded experiments were quantified by counting at least 100 cells per experiment. *P ≤ 0.01 (Student t test).

Fig. 4.

Ape1 associates with TRF2 and POT1 in the cell. (A) Cell cycle-specific association of Ape1 with TRF2. U2OS cells were synchronized by double thymidine treatment and released into fresh medium. Cells were fixed at the indicated time points and immunostained for Ape1 (green) and TRF2 (red). DNA was stained with DAPI. (Scale bar, 5 µm.) (B) Quantification of cell cycle-specific association of Ape1 and TRF2. At least 100 cells per experiment were counted, and cells showing more than three Ape1/TRF2 colocalizing foci were quantified from three independent experiments. *P ≤ 0.01 (Student t test). (C) Ape1 forms a complex with TRF2 in the cell. Nontreated or MMS-treated (15 µg/mL, 16 h) HEK293 cell extracts were subjected to immunoprecipiation with an anti-Ape1 antibody. Immunoprecipitates were then immunoblotted for TRF2 and Ape1. (D) Ape1 colocalizes with POT1 in unperturbed BJ-hTERT cells. Cells were either left nontreated (NT) or treated with MMS (15 µg/mL, 16 h). The cells were then fixed and immunostained for Ape1 (green) and POT1 (red). DNA was stained with DAPI. (Scale bar, 5 µm.) At least 100 cells per experiment were counted, and cells with more than three Ape1/POT1 colocalizing foci were quantified from three independent experiments. *P ≤ 0.01 (Student t test). (E) Mechanism of Ape1 deficiency-associated telomere dysfunction. Our genome is constantly exposed to oxidation as a consequence of routine cell metabolism. Extrinsic factors add further to this load. Upon removal of modified bases from DNA, AP sites form at telomeres and are rapidly processed by Ape1 to direct BER of telomeric lesions. Under Ape1 deficiency, AP sites accumulate at telomeres, causing TRF2 dissociation and telomere uncapping. Unprocessed AP sites are exposed to nuclease attack or converted to DNA breaks during replication. Upon initiation of DNA damage responses, improper repair of damaged telomeres results in telomere fusion and degradation.