The mitochondria are semiautonomous organelles with their genome. The mammalian mitochondrial genome encodes for 13 respiratory chain proteins, 22 tRNAs and 2 rRNAs (Chen and Butow, 2005; Clayton, 1984; Holthöfer et al., 1999). Due to its vital role in oxidative phosphorylation, mitochondrial DNA is frequently exposed to reactive oxygen species (ROS) and is more prone to DNA damage than nuclear DNA, leading to the accumulation of 50 times more mutations, including deletions (Chen et al., 2011; Hudson et al., 1998; Michikawa et al., 1999; Pakendorf and Stoneking, 2005; Yakes and Van Houten, 1997). Mitochondrial mutations have been associated with ageing and several disease conditions like myopathies, dystonia, cancer etc. (Chen et al., 2011; Taylor and Turnbull, 2005). These mutations include point mutations, mismatches, or deletions, which affect the coding of essential proteins involved in oxidative phosphorylation and the respiratory chain (Penta et al., 2001). Mitochondrial mutations, especially large-scale deletions of 200–5000 bp, were associated with the progression of breast, colorectal, renal, and gastric cancers (Bianchi et al., 2001; Carew and Huang, 2002; Modica-Napolitano et al., 2007; Penta et al., 2001). However, the correlation between mtDNA deletions and cancer remains unsettled. The well-studied phenotypes of mtDNA deletions are Kearns-Sayre Syndrome (KSS), Pearson syndrome, and progressive external ophthalmoplegia (PEO; Marni, 2003). Large stretches of deletions seen in different diseased patients suggest fragile sites in the mitochondrial genome similar to the fragile sites (FRA) sites of the nuclear DNA (Richards and Macaulay, 2001).

The FRA regions and other frequent chromosomal translocations have been observed in late replicating sites in the nuclear DNA, which are prone to replication fork stalling due to possible, stable secondary structure formation or inadvertent action of structure-specific nucleases (Dillon et al., 2010; Lin et al., 2013; Raghavan et al., 2004; Sun et al., 2001). Mitochondrial DNA has its replication machinery that is not governed by cell cycle checkpoints. Therefore, it is likely that the formation of a secondary structure may account for observed mitochondrial fragility.

The presence of alternate DNA structures like G-quadruplex, triplex, and cruciform structures in the mitochondrial genome has been reported along with their association with different mitochondrial diseases (Bharti et al., 2014; Damas et al., 2012; Dong et al., 2014; Oliveira et al., 2013). Breakpoint regions reported in the case of PEO patients were in the proximity of G-quadruplex motifs, further suggesting the role of non-B DNA structures in mitochondrial disorders (Bharti et al., 2014). G4 forming motifs were also located adjacent to mitochondrial deletion breakpoints associated with KSS, a clinical subgroup of mitochondrial encephalomyopathies (Van Goethem et al., 2003; Zeviani et al., 1988). Further, a G4 ligand RHPS4 preferentially localises to mitochondria and affects mitochondrial genome content, transcription elongation, and respiratory function (Falabella et al., 2019a; Oliveira et al., 2013). Moreover, in vitro results suggested that a single-base mutation in mtDNA (mt10251) was sufficient to form G4 DNA (Chu et al., 2019). Importantly, ATP-dependent G4 resolving helicase Pif1 could unfold non-B DNA structures and continue the synthesis by mitochondrial DNA polymerases, Pol γ and PrimPol (Butler et al., 2020). However, further studies are required to confirm the presence of these structures in vivo and establish their contribution towards the mechanism of breakage and deletions.

Mitochondrial DNA (mtDNA) 9 bp deletion caused by loss of one copy of the 9 bp repeat sequence (CCCCCTCTA) in the intergenic region of cytochrome c oxidase II (COII)/tRNA^Lys is one of the most common deletions in mitochondria (Redd et al., 1995; Thomas et al., 1998). It has been associated with various diseases, including hepatocellular carcinoma (Jin et al., 2012; Komandur et al., 2011; Krishnan and Turnbull, 2010; Zhuo et al., 2010). The 9 bp deletion is also widely used as a phylogenetic marker to study evolutionary trends and migration of populations (Soodyall et al., 1996; Yao et al., 2000). The occurrence of deletions in such high frequency indicates the fragility of the region; however, its mechanism is unclear.

Out of the large-scale mtDNA deletions reported, one of the well-studied examples is mt⁴⁹⁷⁷, and accumulation of it results in different mitochondrial disorders, including cancer (Yusoff et al., 2019). mt⁴⁹⁷⁷ eliminates mitochondrial sequences between 8470 and 13,447 bp, which include the removal of genes required for normal oxidative phosphorylation (OXPHOS) (Lee et al., 1994; Yang et al., 1994; Yen et al., 1991). Recently the role of replication slippage and DSB repair has been shown to play a role in the generation of mtDNA⁴⁹⁷⁷ in mammalian cells (Phillips et al., 2017). Previous studies have shown that the presence of a 13 bp direct repeat plays a major role in mt⁴⁹⁷⁷, as only one of the repeats was retained following joining, indicating the involvement of microhomology-mediated end joining (Samuels et al., 2004; Srivastava and Moraes, 2005). When restriction endonucleases were used to introduce DSBs, there was the accumulation of deletions, which further implicates the imperfect repair and deletion in the mitochondrial genome (Srivastava and Moraes, 2005). A previous study from our laboratory suggested the existence of microhomology-mediated end joining (MMEJ) in mitochondria, which operate in a microhomology-dependent manner, explaining the frequently seen deletions in mitochondrial DNA (Tadi et al., 2016).

Here, we investigate the mechanism of fragility associated with the most common deletion seen in the mitochondrial genome. The putative role of non-B DNA structures and the mechanism of DNA breakage associated with the formation of mtDNA deletions are extensively studied. We report that the region associated with 9 bp deletion can fold into a G-quadruplex DNA structure using various biochemical and ex vivo methods. For the first time, we show that Endonuclease G can specifically bind to the G-quadruplex structure and cleave the mtDNA. Finally, the joining of the broken DNA could lead to the formation of ‘9 bp deletion’ seen in human mitochondria.

Previously, different non-B DNA motifs, including G4 DNA motifs have been reported in the mitochondrial genome (Dahal et al., 2022; Damas et al., 2012; Dong et al., 2014). It was also suggested that alternate forms of DNA may play a role in the generation of deletions in mitochondria (Dahal et al., 2022; Damas et al., 2012; Dong et al., 2014). In silico studies along with biochemical analyses revealed that 5 classical G4 DNA motifs are present in the mitochondrial genome, which could fold into G4 DNA (Cer et al., 2013; Dahal et al., 2022; Figure 1—figure supplement 1A). Since ‘9 bp deletion’ (8271–8281) is the most frequent mitochondrial deletion and occurs next to one of the 5 G4 motifs with the coordinate position 8252–8295 (designated as mitochondrial Region I) (Figure 1A and B; Dahal et al., 2022), we have investigated the putative role of G4 DNA structure in DNA breakage and formation of the mtDNA deletions. Notably, the chosen G-quadruplex motif is also present in two other large-scale deletions associated with mitochondria.

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EMSA and CD studies to show formation of G-quadruplex structure in Region I.

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Gel shift assay to show abrogation of G-quadruplex structure in mutants of Region I.

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The G4 motif present in Region I of mtDNA can fold into a G-quadruplex structure when present on a plasmid DNA

In the context of double-stranded DNA, G-quadruplexes can form only when the Hoogsteen base pairing between the guanine residues is stronger than the Watson and Crick H-bonding required for duplex B-DNA formation. Therefore, the G-quadruplex forming ability of the mitochondrial Region I was tested after cloning into a plasmid, pDI1. Interestingly, an inverted repeat sequence (coordinate positions 8295–8363), which could fold into a cruciform DNA, was also present adjacent to the G4 motif. Thus, that was also part of pDI1 (Figure 2—figure supplement 1). Further, to delineate the role of G4 DNA in the plasmid context, a mutant plasmid with a scrambled sequence corresponding to the G4 motif but with intact inverted repeats (pDI2) was also constructed (Figure 2A, Figure 2—figure supplement 1). Previous studies have shown that the formation of G4 DNA can lead to a pause during replication (Kumari et al., 2015; Nambiar et al., 2013; Voineagu et al., 2008; Wells, 2009). To assess whether the G4 DNA formed at Region I of the mitochondrial genome can act as a replication pause, a primer extension assay was performed using pDI1 (Kumari et al., 2015; Figure 2B and C). A sequencing ladder was also generated for mapping the location of the polymerase pause sites on the plasmid (Figure 2C). Results revealed multiple polymerase pause sites, and the intensity of the pauses increased with increasing concentrations of KCl (25, 50 and 100 mM; Figure 2C). Interestingly, there was significantly fewer sites and less pausing observed in the absence of KCl, indicating the absence of the formation of intramolecular G-quadruplex species in these cases (Figure 2C, lane 2). Considering that the formation of cruciform DNA occurs irrespective of the presence of KCl, these results also suggest that the observed pause was due to the formation of G4 DNA, but not cruciform DNA. Besides, the observed polymerase pauses mapped to the G-quadruplex motif (Figure 2C, lanes 7–9). Abrogation of G4 DNA structure by mutation of G4 DNA motif (pDI2) resulted in the disappearance of the observed pause site, further confirming the above results (Figure 2D, lanes 3, 4 and lane 6, 7). It was also observed that the appearance of strong polymerase pause sites were dependent on K⁺ (Figure 2E, lanes 4–5; F), but not on other monovalent cations such as Li⁺ (with no or little G-quadruplex stabilising properties) or Na⁺ (with limited stabilising properties) (Figure 2E, lanes 6–9; F) suggesting that the non-B DNA responsible for polymerase arrest was G4 DNA.

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Primer extension studies to show formation of G-quadruplex structure in Region I containing plasmid.

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Mitochondrial Region I can exist as a G-quadruplex structure within the mitochondrial genome and, when present on a plasmid DNA

Formation of G4 DNA on a plasmid or mitochondrial genome could result in single-strandedness at complementary and adjacent regions corresponding to the G4 motif. Sodium bisulphite modification assay was performed on pDI1 to check the single-strandedness in the complementary C strand of Region I. Sodium bisulphite deaminates cytosine resulting in uracil, when present on single-stranded DNA (Figure 3—figure supplement 1A). This change can be read as a C→T conversion after PCR and sequencing (Figure 3—figure supplement 1A, B). Of the 40 clones sequenced, 38 molecules showed C to T conversion to varying extents in either top or bottom strands (Figure 3A; Figure 3—figure supplement 1C). Interestingly, the cytosines complementary to the G-quadruplex motif exhibited single-stranded nature (Figure 3A), suggesting the formation of a G-quadruplex structure in the complementary G-rich strand. Importantly, ~15% of molecules showed continuous conversion at the region corresponding to the G4 motif. These results suggest that Region I of mitochondrial DNA can fold into a non-B DNA even when cloned into a plasmid, consistent with the result obtained following primer extension.

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Sequence of clones after bisulphite treatment in a plasmid containing Region I of mitochondria.

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Sequence of clones after bisulphite treatment in a region I of mitochondria.

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Further, to investigate the existence of G4 DNA structures in the mitochondrial genome, a sodium bisulphite modification assay was performed after extracting the mitochondrial DNA from human cells (Nalm6) under non-denaturing conditions. Results showed that out of 59 DNA molecules analysed following cloning and sequencing, 52 showed C→T conversions to varying extents (Figure 3B and C; Figure 3—figure supplement 1C). Cumulative analysis of converted cytosines on a fragment of 281 bp revealed single-strandedness at Region I corresponding to the complementary region of G4 motifs. When the mitochondrial genome was analysed, regions adjacent to the G4 motif also showed certain levels of single-strandedness (Figure 3B). However, the C→T conversion rate was significantly lower when upstream and downstream sequences were analysed for conversion. Further, among the 50 clones from the top strand, 14 (28%) had continuous conversions of 11 or more cytosines when a stretch of 20 cytosines modifications was considered on a single DNA molecule level (Figure 3C; Figure 3—figure supplement 1C). This suggests that the formation of G4 DNA at Region I may not occur in all DNA molecules of the mitochondrial genome at a given time. However, based on these results in conjunction with biochemical assays, it is evident that the G-quadruplex motif at Region I can indeed fold into a G-quadruplex structure in mitochondria.

BG4, a G-quadruplex binding antibody, binds to G-quadruplex structures in the mitochondrial genome

The previous studies established that BG4 antibody can bind to G-quadruplexes formed in DNA and RNA (Chambers et al., 2015; Das et al., 2016; Javadekar et al., 2020). To study whether BG4 indeed binds to the mitochondrial genome inside cells, immunofluorescence in HeLa and HEK 293T cells was performed after testing the specificity of the antibody (Javadekar et al., 2020). Mito-tracker Deep Red or Mito-tracker Green FM was used for staining mitochondria, while the nucleus was stained with DAPI. BG4 was stained with Alexa fluor-tagged secondary antibodies. Merging of green and red foci resulting in yellow foci was observed in the case of both HeLa and HEK 293T cells, which revealed localisation of BG4 to mitochondria (Figure 4A, Figure 4—figure supplement 1A). The colocalisation was further analysed using the JaCoP plugin in ImageJ software. In both cases, an image with Coste’s mask is presented, showing the extent of localisation in the form of a white dot after merging red and green foci (Figure 4A–C). Colocalisation analyses show Mander’s coefficient value between 0.25–0.4, irrespective of the stain used as MitoTracker, in the case of HeLa and 293T cells indicating that G-quadruplex structures can be detected in the mitochondrial genome in cellular context (Figure 4B and C). To further confirm that the colocalisation was indeed between the MitoTracker and BG4 antibody, immunofluorescence was performed in Rho(0) cells (human osteosarcoma cells with depleted mtDNA) (Dong et al., 2017; Tan et al., 2015). Results suggested that although BG4 foci were observed in the cytoplasm of Rho(0) cells, they do not seem to colocalize with mitochondria (Figure 4D and E).

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Localization of BG4 to mitochondria.

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Immunofluorescence showing the localization of BG4 to mitochondria.

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To examine whether BG4 can indeed bind to mitochondrial G-quadruplexes, in vitro binding of purified BG4 to oligomers harbouring the G-quadruplex region was performed (Figure 4—figure supplement 1B). Results indicated that the bound complex was seen when G-rich oligomer (G1) was incubated with BG4, while such binding was absent when incubated with C-rich oligomer (C1) (Figure 4—figure supplement 1B).

To investigate whether BG4 can bind to G-quadruplex DNA present in the mitochondrial genome, the mito IP-qPCR strategy was employed (Figure 5A, Figure 5—figure supplement 1). Briefly, following crosslinking, mitochondria were isolated, and the region binding to BG4 was immunoprecipitated. Five regions corresponding to G4 motifs and ten random regions without G4 motifs were selected for analysis by using appropriate PCR primers (Figure 5—figure supplement 1). Results showed that all five G-quadruplex forming regions amplified between 10–18 cycles when qPCR was performed (Figure 5B). In contrast, 9 of 10 random regions did not show any amplification following the BG4 pull-down (Figure 5B). Sequence analysis showed that the control region (CR7) amplified after the BG4 pull-down had high GC content. Besides, when CR7 was analysed for potential non-B DNA formation, we noted the presence of two-plate G4 DNA motif. CR7 may likely fold into a 2-plate G4 DNA, which could explain its binding with BG4 (Figure 5B). However, further studies are required to confirm this hypothesis. Samples with input DNA served as a positive control, and as expected, all the regions were amplified. Samples where no antibody was added, did not result in amplification when G4-specific or random region primers were used, revealing the specificity of BG4 towards G4 DNA (Figure 5B). Similar results were also obtained when BG4 pull-down samples were evaluated using semiquantitative PCR (Figure 5C). However, none of the regions amplified when samples from secondary control were used, including CR7 (Figure 5C). Sequence analysis showed that the control region amplified following BG4 pull down had high GC content and predicted for two plates G4 structure, which requires further analysis (Figure 5B and C).

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BG4 ChIP to show the binding of BG4 to mitochondrial G-quadruplex forming regions.

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Thus, our results suggest that Region I and other G4 motifs in the human mitochondrial genome could fold into G-quadruplex DNA. However, based on bisulphite DNA sequencing from Region I, the structure may not be formed in all DNA molecules, and the frequency may be <25%.

Mitochondrial extract induces cleavage at or near the G-quadruplex structure at Region I

To elucidate the mechanism responsible for cleavage at mitochondrial Region I, the involvement of mitochondrial nucleases was considered. To test this, the mitochondrial extract was isolated from the rat tissues (testes and spleen) and used for cleavage assay. The extract was confirmed to be mitochondrial in nature and was tested for cross-contamination by western blotting using the anti-cytochrome C (mitochondrial marker), anti-PCNA (nuclear or cytosolic marker), and anti-Actin (loading control) (Figure 6A). Plasmid harbouring mitochondrial Region I (pDI1) was treated with the mitochondrial extract (0.25, 0.5, 1, and 2 μg) and resolved on an agarose gel following deproteinisation. Interestingly, a concentration-dependent decrease in supercoiled form and increase in the nicked form of plasmid DNA was observed when treated with mitochondrial extracts (Figure 6B, lanes 1–5). Further, there was also a concentration-dependent increase in the linear form of the plasmid DNA, indicating presence of two different nicks at proximity leading to a DSB (Figure 6B, lanes 1–5, C). Although there is a possibility that more than one nick could be present in the pDNA, since the 6 bp mutation in the G4 motif (Figure 6—figure supplement 1A) resulted in abrogation of the conversion of supercoiled to nick DNA, we assume that there will be only a single nick present per strand of the plasmid DNA. To test whether the cleavage observed was due to the presence of G4 DNA, the plasmid bearing mutation in the G4 motif, but bearing intact adjacent inverted repeats (pDI2), was treated with mitochondrial extract and analysed. Interestingly, there was a significant reduction in cleavage when two stretches of G’s were mutated (Figure 6B, lanes 6–10, 6 C; Figure 6—figure supplement 1A), indicating the cleavage was indeed due to the presence of G4 DNA.

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Gel profiles showing the mitochondrial induced cleavage at mitochondrial region I.

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Cleavage assay on plasmid bearing wildtype and mutant G4 sequence.

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For deciphering whether the position of cleavage observed was indeed at Region I, primer extension assay was carried out on pDI1 and its mutant (pDI2 and pDR4; Figure 6—figure supplement 1A) following cleavage by mitochondrial extracts isolated from testes and spleen. Results revealed two specific nicking sites when incubated with mitochondrial extracts from both testes and spleen. These were mapped to the position corresponding to the G-quadruplex forming region when the DNA sequencing ladder was used as a marker (Figure 6D). Interestingly, mutation of the G4 motif resulted in a significant reduction of cleavage activity, as one of the two cleavage sites disappeared when 2 G stretches were mutated (pDI2) from the G4 motif (Figure 6E and F; Figure 6—figure supplement 1). However, the upper cleavage site remains unchanged with the mutation. This may be due to G4 structure formation in alternate conformations, as Region I can fold into multiple forms (Figure 6—figure supplement 2; Dahal et al., 2022). Interestingly, generation of mutation in third stretch of guanines in addition to the above-described mutation (pDR4) resulted in remarkable reduction in the cleavage activity and led to disappearance of both the bands (Figure 6—figure supplement 1). These results confirm the involvement of G4 DNA in the observed cleavage at Region I.

Endonuclease G mediates structure-selective cleavage at mitochondrial Region I

Among different mitochondrial endonucleases, Endonuclease G was shown to bind preferentially to the guanine-rich strands by a previous study (Ruiz-Carrillo and Renaud, 1987). Since we have observed the formation of G-quadruplex DNA structures with multiple conformations (Figure 6—figure supplement 2; Dahal et al., 2022) at Region I of the mitochondrial genome, we considered the possibility of Endonuclease G as the protein responsible for the observed cleavage. In a different study, specific cleavage at kinked DNA by Endonuclease G has also been reported (Ohsato et al., 2002). Based on these observations, we wondered whether Endonuclease G could cleave G4 DNA present at Region I on pDI1. For this, Endonuclease G and its catalytic dead mutant (ΔH151A) were overexpressed in bacteria, purified, and identity was confirmed by western blotting (Figure 7—figure supplement 1A–C). Purified Endonuclease G was subjected to a plasmid nicking assay and analysed on an agarose gel. Results showed that while the wild-type plasmid (pDI1) showed a conformation change from supercoiled to open circular, mutant plasmid (pDI2) retained its supercoiled form (Figure 7A and B). Besides, the linear form of the plasmid DNA was also observed when a higher concentration (60, 90, 120 ng) of Endonuclease G was used for the cleavage in the case of pDI1 (Figure 7A).

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Gel profiles showing the Endonuclease-G-induced cleavage at mitochondrial region I.

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Gel profiles showing the purification of different endonucleases.

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Interestingly, primer extension studies revealed that Endonuclease G cleaved mitochondrial DNA at multiple sites corresponding to a region of G-quadruplex DNA formation at Region I when it was present on pDI1 (Figure 7C, lanes 1–3, 5–8). Importantly, the primary two cleavage positions were comparable to the one observed when the mitochondrial extract was used for the study (Figure 7C, lane 2–4). However, unlike mitochondrial extract, incubation with purified Endonuclease G resulted in cleavage at multiple positions across the G4 motif. It may be that multiple molecules of purified Endonuclease G may bind to different forms of G4 DNA resulting in cleavage at different positions. In the case of mitochondrial extract, additional proteins may bind to G4 DNA, thus preventing cleavage at multiple sites. More importantly, when a protein with a mutation at the nuclease domain of Endonuclease G (ΔH151A) was used, the cleavage activity was abrogated (Figure 7D and E; Figure 7—figure supplement 1B, C). Further, when endonucleases such as CtIP, FEN1 and RAGs were used for cleavage assay, results showed that, unlike Endonuclease G, none of the purified endonucleases exhibited any significant cleavage (Figure 7—figure supplement 1J), although the presence of CtIP (Tadi et al., 2016) and FEN1 (Kalifa et al., 2009; Klungland and Lindahl, 1997; Tadi et al., 2016) has been shown previously in mitochondria. In all cases, the identity and activity of the endonucleases used were confirmed before use (Figure 7—figure supplement 1D–I).

Immunodepletion of Endonuclease G from mitochondrial extracts prepared from rat testes showed a significant reduction in cleavage efficiency at the mitochondrial G-quadruplex Region I, compared to beads alone control (Figure 7F–I). However, immunodepletion of MGME1, another important nuclease known to be present in mitochondria, did not result in any significant difference in the cleavage efficacy (Figure 7G–I; Phillips et al., 2017; Yang et al., 2018). Importantly, the activity was restored when purified Endonuclease G was added back following its immunodepletion from the extract, confirming the role of Endonuclease G in the cleavage (Figure 7J and K).

Endonuclease G is expressed in mitochondria and colocalises with mitochondrial matrix protein TFAM within the cells

Considering that Endonuclease G is one of the essential mitochondrial nucleases (McDermott-Roe et al., 2011; Misic et al., 2016; Wiehe et al., 2018; Zhou et al., 2016), the presence of Endonuclease G was examined in mitochondria, using mitochondrial specific marker, ‘MitoTracker Deep Red or MitoTracker green FM’ and using immunofluorescence with Endonuclease G antibody in three cell lines of different origin (Figure 8A; Figure 8—figure supplement 1). Merge of MitoTracker with tagged Endonuclease G revealed localization of Endonuclease G in mitochondria in HeLa (human cervical cancer), HEK 293T (human embryonic kidney epithelial) and MEF (mouse embryonic fibroblast) cell lines (Figure 8A and B; Figure 8—figure supplement 1). Further, to check if Endonuclease G is present in the mitochondrial matrix, colocalisation analysis was performed along with TFAM, a transcription factor known to be present in the mitochondrial matrix by conducting immunofluorescence studies in HeLa cells (Figure 8C). Results revealed low but distinct levels of colocalisation of Endonuclease G and TFAM, indicating the presence of Endonuclease G in the mitochondrial matrix (Figure 8C and D). Western blotting further confirmed this after fractionation of the mitochondria (see below).

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Localization of Endonuclease G to mitochondria.

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Immunofluorescence showing the Localization of Endonuclease G to mitochondria.

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Further, shRNA-mediated knockdown of Endonuclease G was performed in HeLa cells. Knockdown efficiency was assessed by western blotting using Actin as the loading control (Figure 8E). HeLa cells transfected with scrambled plasmid served as transfection control. Cleavage assay using mitochondrial extracts following knockdown of Endonuclease G revealed a significant reduction in cleavage efficiency at Region I, as that of scrambled control, suggesting its role in mitochondria in the context of cells (Figure 8F and G).

Endonuclease G binds to the G-quadruplex forming regions of mitochondria inside cells

To investigate if Endonuclease G binds to G-quadruplex forming regions of mitochondria, G-quadruplex specific antibody, BG4 was used for colocalisation studies along with anti-Endonuclease G (Figure 9A and B; Figure 9—figure supplement 1A). Yellow foci in the merged image suggested colocalisation of BG4 to Endonuclease G. Approximately 100 cells were analysed for colocalisation of anti-BG4 and anti-Endonuclease G using the Mander’s colocalisation coefficient, for red over green, was ~0.32, while green over red was ~0.21 indicating the binding of Endonuclease G to G-quadruplex DNA structure (Figure 9B).

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ChIP assay showing the binding of Endonuclease G with the mitochondrial G-quadruplex regions within cells.

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ChIP assay showing the binding of Endonuclease G with the mitochondrial G-quadruplex regions when purified Endonuclease G was used.

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P1 nuclease assay showing the binding of Endonuclease G to mitochondrial G quadruples regions.

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An antibody binding assay was performed to examine the binding of Endonuclease G to mitochondrial G quadruplex regions. Purified Endonuclease G was allowed to bind to mitochondrial DNA isolated from Nalm6 cells (Figure 9—figure supplement 1B) and cross-linked. Sonicated mitochondrial DNA was incubated with anti-Endonuclease G and Protein A/G beads. The resulting bound DNA was reverse crosslinked, purified and used for both real-time and semiquantitative PCR (Figure 9—figure supplement 1C, D). The threshold values presented as a bar diagram suggested that Endonuclease G can bind to all G quadruplex regions (GR1- GR5), including the one present at mitochondrial Region I, while the control regions (CR1-CR10) (not known to form canonical G quadruplex) did not bind to Endonuclease G (Figure 9—figure supplement 1C–E). These results reveal the specific binding of Endonuclease G to mitochondrial G quadruplex regions.

Further, the mitochondrial extract was incubated with the mitochondrial DNA, and a pull-down assay was done using Endonuclease G antibody (Figure 9C). Following the pull-down, beads were separated and loaded for protein analysis and western to confirm the pull-down (Figure 9—figure supplement 2A). The remaining was used for DNA isolation, followed by real-time and semiquantitative PCR. Results suggested the binding of Endonuclease G to G-quadruplex regions (Figure 9C–E). Endonuclease G binding assay inside cells was performed to assess the binding of Endonuclease G to mitochondrial G4 DNA (Figure 9F, Figure 9—figure supplement 1C). The antibody-bound mitochondrial DNA was used for real-time PCR to amplify the G quadruplex regions and control regions. Results showed specific amplification of G quadruplex regions and no amplification of control regions, suggesting the specificity of Endonuclease G to G quadruplex regions within the cells (Figure 9F).

P1 nuclease assay was also performed to determine the binding footprint of Endonuclease G to G-quadruplex forming Region I by incubating the wild type and the mutant oligomers of Region I in the presence and absence of Endonuclease G. Interestingly, binding of Endonuclease G to G-rich strand (G1) partially rescued the sensitivity of P1 nuclease (Figure 9—figure supplement 2B, lanes 5–8). In contrast, in the case of C-rich strands, mutants and random oligomers, such protection was absent (Figure 9—figure supplement 2B).

Although Endonuclease G was thought to be localised within the mitochondrial intermembrane space, its proximity to the mitochondrial matrix was supported by multiple experimental evidence suggesting a direct interaction of Endonuclease G with mtDNA (Duguay and Smiley, 2013; McDermott-Roe et al., 2011; Wiehe et al., 2018). Therefore, as a preliminary investigation, we were interested in testing the conditions in which Endonuclease G could be released to the mitochondrial soluble fraction from the inner mitochondrial membrane. To do this, HeLa cells were exposed to Menadione, a mitochondria-specific ROS inducer (25 µM, for 2 hr). Cells were harvested, and fractionation of mitochondria was performed. Fractionated extracts were equalized and used for western blotting studies (Figure 10A). Results showed that exposure to stress conditions resulted in elevated levels of Endonuclease G in the matrix (Figure 10A and B). However, this warrants further detailed investigation. Thus, we hypothesize that different stress conditions may regulate the release of Endonuclease G to mitochondrial matrix.

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Sub localization of Endonuclease G with or without induction of stress.

https://cdn.elifesciences.org/articles/69916/elife-69916-fig10-data1-v3.zip

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Based on the present and previous studies (García-Lepe and Bermúdez-Cruz, 2019; Tadi et al., 2016; Wisnovsky et al., 2018), we propose that under stress conditions, Endonuclease G may be released to the matrix from the inner membrane space where it binds to mitochondrial DNA. This could help the induce DNA breaks by Endonuclease G at Region I of the mitochondrial genome. DNA modifying enzymes such as CtIP, MRN, and Exonuclease G may help in generating 3’ overhangs, following which ligation of broken ends by an unknown mechanism leads to the ‘9 bp deletion’ at the Region I of the mitochondrial genome (Figure 11).

Figure 11

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The present study in conjunction with previous studies, suggest the existence of five G-quadruplex DNA structures in the mitochondrial genome (Dahal et al., 2022; Damas et al., 2012; Dong et al., 2014), all with three-plate conformation that follows the general empirical formula for G-quadruplexes d(G₃₊N_1-7G₃₊N_1-7G₃₊N_1-7G₃₊). G-quadruplexes with three-plate are considered energetically stable (Puig Lombardi and Londoño-Vallejo, 2020). A close analysis of patient breakpoint regions revealed the presence of high-frequency mitochondrial deletion junctions (9 bp deletion) adjacent to one of the G-quadruplex structures at Region I.

The presence of G-quadruplex motifs proximal to mitochondrial deletions has been reported (Falabella et al., 2019a; Falabella et al., 2019b; Oliveira et al., 2013). Computational analysis (Bharti et al., 2014) revealed that G4 sequences proximal to mtDNA deletions in several genetic diseases like Kearns-Sayre syndrome, Pearson marrow-pancreas syndrome, Mitochondrial myopathy, Progressive external ophthalmoplegia, etc. G4 motifs were also located adjacent to mitochondrial deletion breakpoints associated with KSS, a clinical subgroup of mitochondrial encephalomyopathies associated with these diseases (Van Goethem et al., 2003; Zeviani et al., 1988). In the present study, we establish that the region containing ‘9 bp deletion’ can fold into G-quadruplex structures, as shown by EMSA and CD studies in a K+dependent manner. Using bisulphite modification assay, we showed that Region I could fold into a G4 DNA in the mitochondrial genome. Immunofluorescence studies and antibody pull-down assays using G4-specific antibody, BG4, provide further support for the occurrence of G4 quadruplex DNA structures within the cells. Thus, with the combinatorial use of in silico studies, and biophysical, biochemical and ex vivo techniques, we demonstrate the formation of G quadruplexes at the region corresponding to a 9 bp deletion in the mitochondrial genome.

The formation of intramolecular parallel G-quadruplex structure in the mitochondrial genome could make the region fragile as there is an increased probability of replication fork slippage or transcription arrest at this region. The primer extension assay revealed multiple pause sites around the G4 motif, suggesting that polymerase arrest could occur at these structures. However, the deletions seen were precise and occurred in a particular sequence. Thus, an alternate possibility for generating a break at these structures could be due to recognition by structure-selective nucleases followed by cleavage at G4 DNA.

Studies have shown that a few enzymes possess the ability to cleave G-quadruplex structures. The action of RAGs at the G-quadruplex site in BCL2 MBR (Raghavan et al., 2004), MRE11 and DNA2 helicase on either side of the G-quadruplex structure in vitro (Masuda-Sasa et al., 2008) being some of the examples. In the current study, we observed that the structure-selective nuclease activity of mitochondrial endonuclease G could induce cleavage at G4 DNA formed at Region I of the mitochondrial genome using both biochemical (primer extension with purified nucleases, mito-IP with purified Endonuclease G and isolated mitochondria, reconstitution assay with plasmid and purified protein) and ex vivo (immunofluorescence, mito-IP, reconstitution assay in mitochondria) assays. Previous studies have reported various other properties of Endonuclease G, which include the generation of primers during mitochondrial DNA replication by DNA polymerase γ, initiation of genomic inversion in herpes simplex type-1 virus (HSV-1), and its role during apoptosis (Côté et al., 1989; Parrish et al., 2001; Ruiz-Carrillo and Renaud, 1987).

Mammalian Endonuclease G is encoded in the nucleus (Gannavaram et al., 2008; Ohsato et al., 2002; Wiehe et al., 2018; Zhou et al., 2016) and upon translocation to mitochondria, the mitochondrial targeting sequence is cleaved off such that a mature nuclease is released (Li et al., 2001; van Loo et al., 2001; Wiehe et al., 2018). Generally, Endonuclease G is known to reside in the intermembrane space of mitochondria, although a low expression has been reported in the matrix (Duguay and Smiley, 2013; McDermott-Roe et al., 2011; Wiehe et al., 2018). Our preliminary results suggest that Endonuclease G may be further released to the matrix when cells are under stress (Figure 10).

In a recent study, it has been shown that Endonuclease G also cleaved mtDNA in the case of unrepaired oxidative lesions (Wiehe et al., 2018). This was helpful in compensatory mtDNA replication through TWINKLE and mtSSB (Wiehe et al., 2018). However, the binding of Endonuclease G to mtDNA and cleavage might be an event dependent upon various factors, including oxidative stress. Therefore, it is possible that in a typical cellular condition, the level of Endonuclease G in the matrix is low; however, upon stress, it is released into the matrix leading to genome fragility. Hence, the stress-induced release may act as a regulatory mechanism concerning the generation of mitochondrial deletions. A recent study showed the complete degradation property of Endonuclease G at pH 6, which could delineate its role during apoptosis, since apoptotic cells show cytosolic acidification (Schäfer et al., 2004). Interestingly, at physiological pH, the enzyme showed the property of nuclease (converting supercoiled DNA to open circular and linear; Schäfer et al., 2004). CPS-6, a mitochondrial endonuclease G in Caenorhabditis elegans, acts with maternal autophagy and proteasome machinery to promote paternal mitochondrial elimination. It relocates from the intermembrane space of paternal mitochondria to the matrix after fertilisation to degrade mitochondrial DNA (Zhou et al., 2016). Endog^-/- mice revealed that Endonuclease G could act as a novel determinant of maladaptive cardiac hypertrophy, which is associated with mitochondrial dysfunction and depletion (McDermott-Roe et al., 2011).

Our study suggests that Endonuclease G can bind to G-quadruplexes and nick the DNA, resulting in single-strand breaks, which can be converted as double-strand breaks during replication or other DNA transactions. However, in low frequency, we could also see DSBs directly induced by Endonuclease G. We observed that the binding was specific to G4 DNA, as we did not observe nonspecific binding with regions where GC content was high in the mitochondrial genome. Previous studies have shown that although Endonuclease G showed random nuclease activity at higher concentrations, at optimal concentrations, it was specific to kink DNA (Ohsato et al., 2002). The first study on Endonuclease G showed preference towards the guanine-rich strands, which falls in line with our study as the formation of G quadruplexes requires a G-rich region (Ruiz-Carrillo and Renaud, 1987). Further, Endonuclease G cleaved mtDNA in unrepaired oxidative lesions (Wiehe et al., 2018). It is likely that binding and cleavage by Endonuclease G on mtDNA be an event dependent on multiple factors, including oxidative stress. Based on our data, it is evident that in the presence of stress, levels of Endonuclease G go up in the matrix, which enables the protein to be present proximal to the mitochondrial genome. The findings from mito IP studies suggest that Endonuclease G only binds to the G-quadruplex forming regions and not to the other areas of mtDNA. This provides additional regulation and ensures the mitochondrial genome stability in most instances.

DNA repair in mitochondria is poorly understood. Base excision repair (BER) is the most well-characterized DNA repair pathway in mitochondria, while it is generally believed that nucleotide excision repair is absent (Akbari et al., 2008; de Souza-Pinto et al., 2009; Liu et al., 2008). Homologous recombination repair helps repair DSBs and, thus stability of the genome (Dahal et al., 2018), though classical NHEJ was undetectable (Tadi et al., 2016). In contrast, microhomology-mediated end joining uses flanking direct repeats to fix DSBs and delete the sequences between the repeats (Tadi et al., 2016). Interestingly, the G quadruplex forming Region I is close to the deletion junction 8271:8281 (Redd et al., 1995; Yao et al., 2000) also had a 9 bp direct repeat sequence at the flank site.

In a recent study, a replication slippage-mediated mechanism was attributed to a common 4977 deletion where in frequent replication fork stalling at the junction of the common deletion was observed (Phillips et al., 2017). While this is a replication-dependent process, what we observed may not be immediately connected to replication. However, it is possible that replication can indeed facilitate the formation of G-quadruplexes.

A balance between the formation and resolution of G4 DNA within the mitochondrial genome is essential. The mitochondrial genes are transcribed as a single polycistronic mRNA (Clayton, 1984; Sanchez et al., 2011) and therefore undergo a high transcription rate, which may promote G-quadruplex formation. Besides, random ‘breathing’ of DNA (a process of transient melting of the duplex structure due to thermal fluctuations) may also occur in the GC-rich sequences of the mitochondrial DNA promoting G-quadruplex formation (Dornberger et al., 1999; Jose et al., 2009). Once the structure is formed, G4 resolvases also play a critical role in its resolution. In the nuclear DNA context, resolvases like WRN and BLM helicase are considered as the factors that negatively regulate G4 DNA (Giri et al., 2011; Paeschke et al., 2013). However, such a role for these nucleases in mitochondria is not reported. TWINKLE helicase is known to unwind mitochondrial duplex DNA and regulate replication, although its role in structural resolution is yet to be studied (Phillips et al., 2017).

Hence, our study suggests a novel mechanism that explains fragility in mitochondria, dependent on the structure-selective nuclease activity of Endonuclease G on G-quadruplex DNA. The process could be controlled by cellular stress conditions, which may regulate the release of Endonuclease G to the mitochondrial matrix; however, this requires further extensive investigation. Although we have focused on a single example of ‘9 bp deletion’ often seen in mitochondria, it is very much possible that it may be a general mechanism and may help in explaining several other deletions seen in mitochondria-associated human disorders.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data for each data is provided along with the figures.

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Author details

1. Sumedha Dahal

   Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Methodology, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3682-5656

2. Humaira Siddiqua

   Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India

   Contribution

   Formal analysis, Validation, Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

3. Shivangi Sharma

   Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Ravi K Babu

   Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Diksha Rathore

   Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Investigation, Methodology, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

6. Sheetal Sharma

   Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

7. Sathees C Raghavan

   Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Investigation, Methodology, Project administration, Writing – review and editing

   For correspondence

   sathees@iisc.ac.in

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3003-1417

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