Two essential but distinct functions of the mammalian abasic endonuclease

Abstract

The mammalian abasic endonuclease, APE1, has two distinct roles in the repair of oxidative DNA damage and in gene regulation. Here we show that both functions are essential for cell survival. Deletion of the APE1 gene causes embryonic lethality in mice, and no nullizygous embryo fibroblasts have been isolated. We have now established nullizygous embryo fibroblast lines from APE1(-/-) mouse embryos that are transgenic with the "floxed" human APE1 (hAPE1) gene. Removal of hAPE1 by Cre expression through nuclear microinjection elicited apoptosis in these cells within 24 h, which was blocked by coinjection of the wild-type hAPE1 gene. In contrast, mutant hAPE1 alleles, lacking either the DNA repair or acetylation-mediated gene regulatory function, could not prevent apoptosis, although the combination of these two mutants complemented APE deficiency induced by Cre. These results indicate that distinct and separable functions of APE1 are both essential for mammalian cells even in vitro and provide the evidence that mammalian cells, unlike yeast or Escherichia coli, absolutely require APE for survival, presumably to protect against spontaneous oxidative DNA damage.

Fig. 2.

PCR and Southern blot analysis of MEF DNA. (a) PCR primers used for genotyping. Mouse and human APE1 genes are shown with exons (boxes). The primers anneal to both genes, but PCR generates different sized DNA fragments. (b) Representative results of PCR genotyping. Genomic DNA from mouse tails (lanes 1 and 6, normal; lanes 2 and 3, transgenic) or from the –/–tg MEF (lanes 4 and 5) were amplified by using the primers in a. (c) Southern blot analysis by using hAPE1 cDNA as a probe. Genomic DNA from –/–tg (lane 1) or +/–tg (lane 2) MEF were cut with BamHI before Southern hybridization. Filled arrowheads: 1, specific to the hAPE1 transgene; 2, specific to the targeted mAPE1 gene (neoresistance gene insert); 3, specific to normal mAPE1 gene. Open arrow indicates that the bands unaffected by the gene rearrangements. One or two copies of the transgene were integrated in this transgenic mouse and MEF strain, based on band intensity in the Southern analysis.

Fig. 4.

Caspase 3 activation after APE1 removal by Cre expression. (a and b) Bicistronic vectors for simultaneous expression of Cre and humanized recombinant GFP (hrGFP) proteins from a single pCMV promoter. (a) The cre gene was inserted into a phrGFP vector (Stratagene). An internal loxP site in the vector was replaced with a PacI site, which was then used to insert the WT hAPE1 cDNA with pCMV in b. Plasmids expressing Cre and hrGFP proteins (c–e), or expressing WT APE1 in addition to Cre and hrGFP (f–h) were used for transient transfection. After 36 h, the cells were fixed and stained with anti-Cre, anti-APE1, and anti-activated caspase 3 antibodies, followed by rhodamine-conjugated secondary rabbit antibody. G, GFP; R, rhodamine; T, transmission; M, merge. The white arrow in h denotes a dead cell that was untransfected (GFP negative) but caspase 3-positive.

Fig. 1.

Generation of conditional Ape1 null mouse. (a) Construction of transgenic mice expressing hAPE1. (Left) A 6-kb human APE1 genomic DNA with intrinsic HindIII (upstream) and XbaI (downstream) sites was cloned into pBluescript SK(–) (Stratagene) and loxP elements were inserted at both ends. (Center) Expression of APE1 was confirmed with Northern and Western blotting, showing 2- to 3-fold higher expression in transgenic mouse livers. (Right) A typical Southern blot for genotyping transgenic mice after BamHI digestion and probing with hAPE1 cDNA. Filled arrowhead, transgene-specific band; open arrowhead, specific to the neointegrated mAPE1 gene. (b) Genotyping of progeny mice after intercrossing (+/–; tg). Numbers at the bottom indicate occurrence of the corresponding genotypes. With the total number of mice tested (109), the probability of finding no –/–tg mouse based on Mendelian law is <1 × 10^–12.

Fig. 3.

Effect of APE1 deletion on cell survival. (a–e) Cre expression vector was coinjected into nuclei with Oregon Green and a vector control (a and c–e) or a WT APE1 expression vector (b). Two representative results are shown in a and b, with an inset for divided cells (b). PC, phase contrast; OG, Oregon Green. The cells were photographed at 24 h after the injection (a and b) or at indicated times (c–e). The arrow in e indicates cells in which the cre plasmid was injected into the cytoplasm, which appeared normal and divided after 24 h. (f and g) Cell death induced by expression of Cre-EGFP fusion protein. The –/–tg MEF (f) or BALB/c 3T3 control cells (g) were transfected with the Cre-EGFP vector. Cells were stained with propidium iodide without fixation, and then fluorescence of EGFP (G) and propidium iodide (P) was visualized with confocal microscopy. Arrowheads indicate sites of chromatin condensation. T, transmission; M, merged.

Fig. 5.

Mutational analysis of the protective effect of APE1. Survival analysis of MEF after microinjection with K6R/K7R (a), H309N (b), or K6R/K7R + H309N (c) plasmids and the Cre plasmid and Oregon Green. (d) Bar graph of cells with various morphologies related to apoptotic response, as described in the figure, which were visually assessed at 24 h after microinjection (when Oregon Green was visible even after mitosis). The experiments were carried out three to nine times (>60 total cell numbers for each APE1 cDNA). Error bars denote standard deviations.