Regulation of base excision repair: Ntg1 nuclear and mitochondrial dynamic localization in response to genotoxic stress

Abstract

Numerous human pathologies result from unrepaired oxidative DNA damage. Base excision repair (BER) is responsible for the repair of oxidative DNA damage that occurs in both nuclei and mitochondria. Despite the importance of BER in maintaining genomic stability, knowledge concerning the regulation of this evolutionarily conserved repair pathway is almost nonexistent. The Saccharomyces cerevisiae BER protein, Ntg1, relocalizes to organelles containing elevated oxidative DNA damage, indicating a novel mechanism of regulation for BER. We propose that dynamic localization of BER proteins is modulated by constituents of stress response pathways. In an effort to mechanistically define these regulatory components, the elements necessary for nuclear and mitochondrial localization of Ntg1 were identified, including a bipartite classical nuclear localization signal, a mitochondrial matrix targeting sequence and the classical nuclear protein import machinery. Our results define a major regulatory system for BER which when compromised, confers a mutator phenotype and sensitizes cells to the cytotoxic effects of DNA damage.

Figure 1.

Definition of functional intracellular targeting signals within Ntg1. (A) Schematic of Ntg1. Predicted critical residues for nuclear and mitochondrial localization and catalytic activity of Ntg1 are indicated, including the putative MTS (residues 1–26), two putative cNLSs (residues 14–16 and 31–37) and the putative active site (residues 233–245). Amino acids depicted in green were altered in this study in order to examine Ntg1 function (Table 1). (B) The localization of GFP-tagged Ntg1 proteins (Supplementary Table S1) was assessed via direct fluorescence microscopy. GFP (green), DAPI (blue), Mitotracker (red) and merged images of cells expressing wild-type Ntg1 (WT), Ntg1_nls1, Ntg1_nls2, Ntg1_nls1/2 and Ntg1_mts variants of Ntg1-GFP are shown. (C) Quantification of localization of Ntg1-GFP variants to nuclei only (nuclear), mitochondria only (mito), or nuclei plus mitochondria (nuc + mito) was determined for each cell examined and plotted as percentage of the total cells evaluated for at least 200 cells per variant. Error bars represent standard deviation. (D) Quantification of steady-state expression levels of Ntg1 variants by immunoblotting and densitometry. Five separate experiments were quantified. The expression of Ntg1-GFP was normalized and the mean level of wild-type Ntg1-GFP was set to 1.0. Error bars represent standard deviation.

Figure 2.

The bipartite cNLS of Ntg1 is sufficient to direct nuclear localization of Ntg1. GFP (green), DAPI (blue) and merged images of untreated cells expressing Ntg1_cNLS1-GFP₂, Ntg1_cNLS2-GFP₂, Ntg1_cNLS1/2-GFP₂ and two proteins, the negative control GFP₂ alone (empty vector) and the bipartite positive control SV40_cNLS-GFP₂ (34).

Figure 3.

The classical nuclear protein import pathway is required for nuclear localization of Ntg1. (A) Localization of Ntg1-GFP (Ntg1), and two control proteins, a cNLS cargo, SV40 bipartite cNLS (cNLS) (34) and a non-cNLS cargo (68), Nab2-GFP (Nab2), was assessed via direct fluorescence microscopy in untreated cells. GFP (green), DAPI (blue) and merged images of wild-type (WT), importin α (srp1-54), and control sxm1Δ mutant cells are shown at the nonpermissive temperature, 37°C. See also Supplementary Figure S1. (B) An in vitro binding assay reveals direct binding of Ntg1 to the cNLS receptor, importin-α. Either GST-ΔIBB-importin-α (Imp-α) or GST alone (GST) as a control was incubated with Ntg1-His₆ or a His-tagged control protein Nab2-His₆ as described in ‘Materials and Methods’ section. Both the unbound (U) and bound (B) fractions were resolved by SDS-PAGE and then stained with Coomassie blue. Albumin, which was used as a nonspecific protein competitor, is present in the unbound fractions.

Figure 4.

Functional intracellular targeting signals are required for dynamic localization of Ntg1 in response to oxidative DNA damage. Quantification of Ntg1-GFP, Ntg1_nls2-GFP and Ntg1_mts-GFP localization following cellular stress. Cells were not treated (NT) or were exposed to 20 mM H₂O₂, 55 mM MMS, or 20 mM H₂O₂ plus 10 µg/ml antimycin (HA). The localization of Ntg1-GFP variants to nuclei only (nuclear), mitochondria only (mito) or nuclei plus mitochondria (nuc + mito) was determined for each cell and plotted as percentage of the total cells evaluated for at least 100 cells per variant and condition. Error bars represent standard deviation. WT-HA and WT-MMS nuc + mito standard deviations are small and are obscured by the data point symbols.

Figure 5.

Functional analysis of the dynamic localization of Ntg1. (A) The H₂O₂ sensitivity of wild type (WT), apn1 ntg2 ntg1 rad1 (BER⁻/NER⁻), apn1 ntg2 NTG1 rad1 (BER*_wt/NER⁻) and apn1 ntg2 ntg1_mutant rad1 (BER*_mutant/NER⁻) cells were assessed. The percent survival was set to 100% for untreated samples and was determined for 0, 2, 4 and 6 mM H₂O₂ doses. (B) The MMS sensitivity of wild type (WT), (BER⁻/NER⁻), (BER*_wt/NER⁻) and (BER*_mutant/NER⁻) cells were assessed. The percent survival was set to 100% for untreated samples and was determined for 0, 1, 3 and 5 mM MMS doses. Error bars indicate standard deviations in data.

Figure 6.

Amino acid substitutions within intracellular targeting signals do not affect the catalytic activity of Ntg1. Ntg1 DNA glycosylase/AP lyase activity was assessed by monitoring cleavage of a ³²P 5′-end-labeled oligonucleotide (31mer) containing dihydrouracil by the Ntg1 variant proteins, Ntg1, Ntg1_nls1, Ntg1_nls2, Ntg1_mts or Ntg1_catalytic. The positions of the uncleaved 31-mer oligonucleotide (UC) and the cleaved 13-mer oligonucleotide (CP) are indicated. No enzyme was added to the negative control lane (-Ctrl). Protein concentrations are as follows from left to right: 1.85 ng/μl, 5.5 ng/μl, 16.6 ng/μl and 50 ng/μl. All lanes are from the same gel at the same exposure, the black line represents lanes that were cropped from the image.

Figure 7.

Model of Ntg1 dynamic localization in response to nuclear and mitochondrial oxidative DNA damage. Nuclear oxidative DNA damage signals (NODDS) and mitochondrial oxidative DNA damage signals (MODDS) compete for the recruitment of Ntg1 to sites of oxidative DNA damage from the cellular pool of Ntg1. The cellular pool is comprised of Ntg1 in constant flux between the nucleus and the cytoplasm. NODDS (blue) promote the association between Ntg1 and DNA damage responders in the cell. These responders are members of networks of stress response pathways. The classical nuclear protein import machinery, including importin α/β, is one such DNA damage responder that is activated by NODDS. MODDS (red) activate another class of DNA damage responders. The response to NODDS and MODDS by Ntg1 and DNA damage responders results in appropriate concentrations of Ntg1 in nuclei and mitochondria, given the oxidative DNA damage levels in each organelle. Once localized to these organelles, Ntg1 facilitates the repair of nuclear or mitochondrial oxidative DNA damage, thus preventing cell death and promoting genomic stability. Black arrows represent localization under steady-state conditions.