Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Abstract

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3' blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

Figure 1

APE activity of Heparin–Sepharose fractions. (A) APE activity of fractions was measured at 37°C for 10 min with 1:200 diluted fractions and 500 nM substrate. (B) Quantitative representation of APE activity in active fractions (10–16) after 1:500 dilution.

Figure 2

Identification of purified bovine mtAPE. (A) Coomassie staining of fraction 12 (25 µl; Figure 1). Lane 1, Recombinant NΔ33 hAPE1. Lane 2, fraction 12; M, molecular weight markers. (B) Western blot of fraction 12 with APE1 antibody. Lane 1, 20 ng recombinant hAPE1; Lane 2, fraction 12 (2 µl); Lane 3, recombinant NΔ20 APE1 (25 ng).

Figure 3

Confirmation of mtAPE as NΔ33 APE1. (A) MS analysis of trypsin-digested mtAPE band (fraction 12). (B) N-terminal sequence of mtAPE of EKEAV (shown in the box).

Figure 4

Kinetic parameters for recombinant hAPE1 (closed square) and bovine mtAPE (closed triangle). Dependence on (A) pH, (B) Mg²⁺ and (C) KCl.

Figure 5

Comparative endonuclease activity of recombinant hAPE1 and bovine mtAPE (fraction 12). (A) Quantification of hAPE1 and bovine mtAPE from western analysis. Lanes 1–3, 4, 8 and 16 ng of APE1, respectively; lanes 4–6, 0.5, 1 and 2 µl of fraction 12, respectively. (B) THF-oligo substrate was incubated with hAPE1 or fraction 12 as described in Materials and Methods. Lane 1, no enzyme; lane 2, 1 fmol hAPE1; lane 3, 0.5 fmol hAPE1; lanes 4–5, fraction 12, at 1000- and 2500-fold dilution, respectively.

Figure 6

Relative activity of NΔ33 and full-length hAPE1. (A) Endonuclease activity, lane 1, control; lanes 2–4, 0.1, 0.2 and 0.5 fmol full-length APE1; lanes 5–7, 0.1, 0.2 and 0.5 fmol NΔ33 APE1. (C) 3′ Phosphodiesterase activity, lane 1, control; lanes 2, 4 and 6; 100, 200 and 300 fmol APE1; lanes 3, 5and 7; 100, 200 and 300 fmol NΔ33 APE1. (B and D) Graphical representation of the results in (A) and (C), respectively.

Figure 7

Presence of NΔ33 APE1 in beef liver mitochondria. (A) Lane 1, full-length and NΔ33 APE1 markers; lane 2, mitochondrial extract (50 µg); lane 3, extract (50 µg) of sucrose density gradient-purified mitochondria; lane 4, 25 µg extract after trypsin treatment for 20 min. (B) Lane 1, APE1 and NΔ33 APE1 markers; lanes 2–4, extract (25 µg) of nucleus, cytoplasm and mitochondria, respectively.

Figure 8

Presence of NΔ33 APE1 in NIH3T3 cells. (A) Western analysis. Lane 1, recombinant full length hAPE1; lane 2, 20 µg nuclear extract; lane 3, 20 µg cytoplasmic fraction; lane 4, 50 µg mitochondrial extract; lane 5, 50 µg mitochondrial extract after trypsin treatment for 20 min; lane 6, recombinant NΔ33 APE1. (B) Specific cleavage of full-length ³⁵S-labeled hAPE1 by mitochondrial extract. Lane 1, APE1 control; lanes 2–4, treatment with mitochondrial, nuclear and cytoplasmic extracts, respectively as described in Materials and Methods. Full-length and cleaved products in duplicates (a and b) are indicated by arrows.

Figure 9

Intracellular localization of full-length and truncated hAPE1 with C-terminal EGFP tag. (A) Nuclear as well as cytoplasmic and mitochondrial localization of full-length APE1-EGFP in live cells. (B) Nuclear localization of full-length APE1 in fixed cells with nuclear DAPI staining. (C and D) Colocalization of NΔ20 APE1-EGFP and NΔ41 APE1-EGFP with MitoTracker Red showing their presence in the mitochondria.