Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3'-phosphatase in maintenance of mitochondrial genome

Abstract

The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3'-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3'-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.

FIGURE 1.

Identification of NEIL2 and PNKP in mitochondria. A, a 5′-³²P-labeled 51-mer oligo (5-OHU·B11; Table 1A) was used for a DNA glycosylase/AP lyase assay with purified ME (10 μg; lane 2). Lane 1, no protein; lane 3, purified NEIL2 (20 fmol) as a positive control. B, trapping assay of purified NEIL1 (20 fmol; lane 2), NEIL2 (lane 3), and ME (10 μg; lane 4) with 5′-³²P-labeled 5-OHU·B11 oligo. Trapped complexes of the NEILs and free DNA are indicated. C, Western analysis of NEIL2 depletion in HEK293 cell extracts (10 μg) by NEIL2-specific siRNA; tubulin was used as a loading control. D, DNA glycosylase/AP lyase activity (with 5′-³²P-labeled 5-OHU·B11) in the ME (5 μg) prepared from control (lane 2) versus NEIL2-depleted (lane 3) cells. E, Western blot analysis of cytosolic, nuclear, and mt fractions from HEK293 cells. Abs specific for RNAP II, lactate dehydrogenase (LDH), and the 70-kDa subunit of the electron transport chain complex II (C II-70kDa) were used as nuclear, cytosolic, and mt markers, respectively, to show the purity of the mt preparation. Cytosolic, nuclear, and mitochondrial fractions were loaded in equal amounts (30 μg). Purified NEIL2 and PNKP (10 ng) were used as references. S, ³²P-labeled 3′-P-containing oligo substrate; P, released phosphate; siRNA-C, siRNA control.

FIGURE 2.

Colocalization of NEIL2 and PNKP in mitochondria. The mitochondrial presence of NEIL2 (A) and PNKP (B) was investigated in human neuroblastoma SH-SY5Y cells using Abs for NEIL2 (rabbit polyclonal; see Ref. 5), PNKP (rabbit polyclonal), and mitochondrion-specific MT-CO2 (mouse monoclonal; Santa Cruz Biotechnology). The nuclei are counterstained with DAPI (blue). Red, NEIL2 and PNKP; green, Mitochondria; yellow, overlap of red and green showing significant co-localization of NEIL2 and PNKP with MT-CO2. Other details are under “Experimental Procedures.”

FIGURE 3.

A, ChIP assay. After sonication and immunoprecipitation of cross-linked chromatin separately with Ab for FLAG (panel i), PNKP (panel ii), or RNAP II (panel iii), the IPs were washed, the bound protein-DNA complexes were eluted, and the precipitated DNA was amplified by PCR using mitochondrial (MT-CO2 and MT-CO3) or nuclear (β-actin) gene-specific primers. B, re-ChIP assay. The bound fractions from the first IP were eluted, divided into two aliquots, and subjected to a second IP with IgG (as control) or with a specific Ab. PCR amplifications were performed using specific primers as shown in Table 1B.

FIGURE 4.

Detection of NEIL2 and PNKP (mouse Ab) interaction with Polγ (rabbit Ab) in HEK293 cells by proximity ligation assays. Upper panel, PNKP (mouse monoclonal; a gift from Dr. Michael Weinfeld) with Polγ (anti-POLG1; Agrisera AB) or IgG (rabbit Ab). Lower panel, NEIL2 (mouse monoclonal; Abnova) with Polγ (rabbit Ab) or IgG (rabbit Ab).

FIGURE 5.

Representative gel showing 3′-phosphatase activity of PNKP in mitochondria. The substrate for the PNKP 3′-phosphatase assays was generated as we have described previously (7). A, Western analysis of PNKP in control (lane 1) and PNKP-specific miRNA-expressing cells (lane 2). Upper panel, RNAP II as control. PNKP transcript levels in control versus miRNA-treated cells were quantitated by quantitative PCR (right panel). B, a ³²P-labeled 3′-phosphate-containing oligo (0.5 pmol) was used to measure the 3′-phosphatase activity of PNKP in MEs (5 and 10 μg) prepared from control (Con) miRNA-expressing (lanes 6 and 7) and PNKP-depleted cells (lanes 4 and 5). Lane 1, substrate alone; lanes 2 and 3, purified PNKP (50 and 100 fmol) as a positive control. S, ³²P-labeled 3′-P-containing oligo substrate; P, released phosphate. The histogram shows quantitation of the percentage (%) of product (³²P_i) released. The standard error of the mean was calculated from at least three independent experiments. The radioactive bands were quantitated using Quantity One software from Bio-Rad. S.E. was calculated using Microsoft Excel 7.0.

FIGURE 6.

Representative gel showing BER and SSBR using mt extract. A, efficient repair of a 5-OHU lesion in a duplex oligo (top) measured by incorporation of [α-³²P]dCMP after excision of 5-OHU. Lane 1, no protein; lanes 2 and 3, ME from control (Con) cells (5 and 10 μg); lanes 4 and 5, PNKP-depleted ME (5 and 10 μg). Addition of purified PNKP (50 and 100 fmol) restored efficient repair (lanes 6 and 7). Lane 8, size markers. The histogram (bottom) represents quantitation of the repair products with lane 2 arbitrarily set as 1. B, a plasmid DNA containing a single U was treated with Udg/Fpg to generate 3′-P and 5′-P with a one nucleotide gap to assess the 3′-phosphatase activity of PNKP. Repair was then monitored by incorporation of [α-³²P]dCMP in the presence of Polγ and LigIIIα. The repaired product was digested with N.BstNBI and analyzed by denaturing gel electrophoresis. Other details are provided under “Experimental Procedures.” Lane 1, purified Polγ and LigIIIα (50 fmol); lane 2, reconstitution of SSBR using purified PNKP, Polγ, and LigIII (50 fmol each); lanes 3 and 4, ME (5 and 10 μg); lanes 5 and 6, PNKP-depleted ME (5 and 10 μg); lanes 7 and 8, PNKP-depleted ME plus purified PNKP (50 and 100 fmol); lane 9, size markers. Quantitation of the radioactive bands (lanes 2–8) is represented in a histogram (bottom) with lane 3 arbitrarily set as 1. A schematic representation of the generation a 3′-P-containing plasmid DNA substrate is shown (top). The standard error of the mean was calculated from at least three independent experiments.

FIGURE 7.

Quantitation of mitochondrial DNA damage in NEIL2- and PNKP-depleted cells. Upper panel, representative gel showing PCR-amplified fragments of the long amplicon (8.9-kb region) and 211-bp region. The relative levels of endogenous DNA damage were calculated by quantitating the long amplicon (8.9-kb region) PCR product after normalizing for mt copy number by PCR of a 211-bp region of mt genome. Bottom panel, quantitation of the amplified products is represented in histograms with the amplicon from control siRNA- or miRNA-treated cells arbitrarily set as 100. The DNA from NEIL2-depleted cells was digested with Fpg/endonuclease III before analysis. Other details are provided under “Experimental Procedures.” *, p < 0.01. The standard error of the mean was calculated from at least three independent experiments.