Human EXOG Possesses Strong AP Hydrolysis Activity: Implication on Mitochondrial DNA Base Excision Repair

Abstract

Most oxidative damage on mitochondrial DNA is corrected by the base excision repair (BER) pathway. However, the enzyme that catalyzes the rate-limiting reaction─deoxyribose phosphate (dRP) removal─in the multienzymatic reaction pathway has not been completely determined in mitochondria. Also unclear is how a logical order of enzymatic reactions is ensured. Here, we present structural and enzymatic studies showing that human mitochondrial EXOG (hEXOG) exhibits strong 5'-dRP removal ability. We show that, unlike the canonical dRP lyases that act on a single substrate, hEXOG functions on a variety of abasic sites, including 5'-dRP, its oxidized product deoxyribonolactone (dL), and the stable synthetic analogue tetrahydrofuran (THF). We determined crystal structures of hEXOG complexed with a THF-containing DNA and with a partial gapped DNA to 2.9 and 2.1 Å resolutions, respectively. The structures illustrate that hEXOG uses a controlled 5'-exonuclease activity to cleave the third phosphodiester bond away from the 5'-abasic site. This study provides a structural basis for hEXOG's broad spectrum of substrates. Further, we show that hEXOG can set the order of BER reactions by generating an ideal substrate for the subsequent reaction in BER and inhibit off-pathway reactions.

Figure 1.

AP removal activity of hEXOG, Pol β, and Pol γ. Activities of hEXOG (blue), Pol β (green), and Pol γ (red) on digestion of 5′-³²P-labeled DNA dRP (a) or THF (b). For clarity, the biotin blockers on the DNA ends are not shown. The graphs represent the mean values with standard deviations (error bars) from three experiments. Reactions were carried out for 5 min at 37 °C using 5 nM DNA substrate and hEXOG, Pol β, or Pol γ in the presence of 10 mM MgCl₂.The raw data are presented in Figure S1.

Figure 2.

hEXOG cleavage of the oxidized abasic site dL. (a) Conversion of nitroindole (NI)-containing DNA to deoxyribonolactone (dL) and the hydrolysis activity of hEXOG on dL-containing DNA. Lane 1: molecular weight standard, lane 2: NI-DNA, lane 3: NaOH-treated NI-DNA, lane 4: UV-treated NI-DNA, lane 5: UV- and then NaOH-treated NI-DNA, lanes 6–9: dL-containing DNA (10 nM) processed by hEXOG for 5 min at 37 °C in the presence of 10 mM MgCl₂. (b) Quantification of the data in panel (a). The graph presents the mean values with deviations (error bars) from two experiments. (c) Schematic of dL-containing DNA synthesis.

Figure 3.

dRP removal by hEXOG and Pol β. (a) hEXOG (51 nM) or Pol β (58 nM) incubated at 0 °C with 10 mM 5′-³²P-dRP-containing DNA and 20 mM NaBH₄ with or without 10 mM MgCl₂. (b) Quantification of enzyme concentration-dependent dRP removal (raw data are shown in Figure S4).

Figure 4.

Crystal structures of hEXOG H140A. (a) Active site of hEXOG shows the scissile bond at the third phosphodiester bond, yielding a 5′-THF-2 nt product. The catalytic H¹⁴⁰ is docked from the Apo hEXOG structure after superposition of the Core domains. The electron density of THF-10 nt/11 nt DNA is shown in the blue mesh. F³⁰⁷ contributes to the tape-measured cleavage. 5′-THF is stabilized by R³¹⁴, K¹⁴⁸, and Y³¹⁰ from the Wing domain. (b) Overall structure showing the homodimer hEXOG; each monomer contains a catalytic Core domain (green) and a Wing domain (gray). The DNA contains a 5′-THF-10 nt substrate (magenta) and an 11 nt complementary strand (gray). (c) Scheme of the DNA substrate. (d) Proposed nucleolytic reaction mechanism of hEXOG. (e) Interaction of THF with hEXOG. (f) Terminal base pair interaction with F307, Y310, and L311.

Figure 5.

Crystal structure of a partially gapped DNA and the effect of hEXOG on the order of BER reactions. (a) Active site of 10/12 nt partial duplex showing the strand separation by K-helix of the Core domain and Y³¹⁰ and F³⁰⁷ of the Wing domain. The −1 and −2 residues of the complementary strand are flipped out by ~90°. The DNA sequence in the crystal is shown. (b) Surface rendition illustrating that the 3′-end of the complementary strand departs from the 5′-end of the substrate strand. (c) Modeling indicates that the 5′-end of the substrate strand is protected by hEXOG. (d) Ligation of the THF-containing substrate (50 nM) in the presence of Lig3 (50 nM) and increasing concentrations (10, 25, 50, 100, 200 nM) of hEXOG. Reactions were carried out for 5 min at 37 °C in the presence of 10 mM MgCl₂ and 1 mM ATP. A representative image from three independent experiments is shown.

Scheme 1.. BER in Nucleus and in Mitochondria^a

^aThe nuclear BER is based on existing literature, and the mitochondrial pathway is proposed based on this study.