Unleashing a novel function of Endonuclease G in mitochondrial genome instability
- Department of Biochemistry, Indian Institute of Science Bangalore, India
- Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, India
Figures
Figure 1 with 1 supplement
Biochemical studies to investigate formation of G-quadruplex structure in Region I of the mitochondrial genome.
(A) Sequence of the mitochondrial Region I with direct repeats and inverted repeats. Blue-shaded arrows represent the direct repeats, and yellow arrows represent the inverted repeats. The sequence …
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Figure 1—source data 1
EMSA and CD studies to show formation of G-quadruplex structure in Region I.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig1-data1-v3.zip
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Figure 1—source data 2
Gel shift assay to show abrogation of G-quadruplex structure in mutants of Region I.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig1-data2-v3.zip
Figure 1—figure supplement 1
Evaluation of G-quadruplex formation at mitochondrial Region I.
Related to Figure 1. (A) Schematic representation of mitochondrial genome showing positions of five G-quadruplex DNA motifs. (B) List of oligomers used for studying effect of mutation of G stretches …
Figure 2 with 1 supplement
Evaluation of formation of G-quadruplex DNA in Region I of the mitochondrial DNA when cloned into a plasmid.
(A) Schematic showing cloning of the mitochondrial Region I and its mutant to generate plasmids pDI1 and pDI2, respectively. The duplex region containing the G stretches is depicted in blue, while …
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Figure 2—source data 1
Primer extension studies to show formation of G-quadruplex structure in Region I containing plasmid.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig2-data1-v3.zip
Figure 2—figure supplement 1
Schematic showing cloning of the mitochondrial Region I and its mutant to generate plasmids, pDI1 and pDI2.
Related to Figure 2. The duplex region containing the G stretches are depicted in blue, while the mutated nucleotides are marked in red. A 68 nucleotide sequence is also shown with inverted repeats …
Figure 3 with 1 supplement
Evaluation of G-quadruplex formation at mitochondrial Region I.
(A) Bisulphite modification assay on plasmid (pDI1) containing mitochondrial Region I. Vertical bar represents the number of times the respective cytosine in the top and bottom strands of …
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Figure 3—source data 1
Sequence of clones after bisulphite treatment in a plasmid containing Region I of mitochondria.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig3-data1-v3.zip
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Figure 3—source data 2
Sequence of clones after bisulphite treatment in a region I of mitochondria.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig3-data2-v3.zip
Figure 3—figure supplement 1
Schematic showing conversion of cytosine to thymine following bisulfite modification assay.
Related to Figure 3. (A, B) Treatment with sodium bisulfite can result in deamination of cytosine, leading to uracil when present on a single-stranded DNA (A). This C to U conversion can lead to C …
Figure 4 with 1 supplement
Evaluation of G-quadruplex structure in mitochondria of cells.
(A) Representative images of HeLa and HEK293T cells showing colocalisation of BG4, the G4 binding antibody to mitochondria following immunofluorescence assay. The nucleus is stained with DAPI (blue …
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Figure 4—source data 1
Localization of BG4 to mitochondria.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig4-data1-v3.zip
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Figure 4—source data 2
Immunofluorescence showing the localization of BG4 to mitochondria.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig4-data2-v3.zip
Figure 4—figure supplement 1
Evaluation of existence of G-quadruplex in mitochondria of cells.
Related to Figure 4. (A) Immunofluorescence study showing colocalization of BG4 (antibody that binds to G4 DNA) and mitochondrial genome. Nucleus from HeLa cells is stained with DAPI (blue color). …
Figure 5 with 1 supplement
Evaluation of BG4 binding to G-quadruplex structure in mitochondrial genome.
(A) Schematic showing the experimental strategy used for mito-IP using anti-BG4. Briefly, cells were crosslinked and then mitochondria were isolated and sonicated to obtain the small fragments of …
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Figure 5—source data 1
BG4 ChIP to show the binding of BG4 to mitochondrial G-quadruplex forming regions.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig5-data1-v3.zip
Figure 5—figure supplement 1
Evaluation of existence of G-quadruplex in mitochondrial DNA.
Related to Figure 5. Schematic showing the position of primers used for mito IP studies. GR1-GR5 represents primers that can amplify G-quadruplex forming motifs and CR1-CR10 represents the random …
Figure 6 with 2 supplements
Evaluation of mitochondrial extract induced cleavage at G4 DNA formed at Region I of the mitochondrial genome.
(A) Purification and characterization of mitochondrial extracts. Mitochondrial extract was prepared from rat testis and spleen, and its purity was evaluated using specific markers by western …
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Figure 6—source data 1
Gel profiles showing the mitochondrial induced cleavage at mitochondrial region I.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig6-data1-v3.zip
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Figure 6—source data 2
Cleavage assay on plasmid bearing wildtype and mutant G4 sequence.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig6-data2-v3.zip
Figure 6—figure supplement 1
Mitochondrial extract mediated cleavage assay on plasmids bearing different mutations at G4 DNA motif containing plasmid.
Related to Figure 6. (A) Table showing the positions of different mutations at G4 DNA motif on plasmid bearing Region I. (B) Cleavage assay using different mutations at G4 DNA motif on plasmid …
Figure 6—figure supplement 2
DNA sequences that support formation of different conformation of G-quadruplex DNA structures in Region I.
Related to Figure 6. The presence of five G-stretches (shown in blue) and three GNG stretches (shown in green) allows this region to fold into four different conformations of G-quadruplex structure.
Figure 7 with 1 supplement
Studies to identify mitochondrial nuclease responsible for cleavage at G4 DNA formed at Region I of the mitochondrial genome.
(A) In vitro nicking assay using purified Endonuclease G on wild type and mutant plasmids containing mitochondrial Region I (pDI1 and pDI2). Both wild type and mutant plasmids were treated with …
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Figure 7—source data 1
Gel profiles showing the Endonuclease-G-induced cleavage at mitochondrial region I.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig7-data1-v3.zip
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Figure 7—source data 2
Gel profiles showing the purification of different endonucleases.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig7-data2-v3.zip
Figure 7—figure supplement 1
Overexpression, purification and activity assay of different endonucleases.
Related to Figure 7. (A, B) Overexpression and purification of Endonuclease G protein (wild type and mutant). The purity and identity of the protein was confirmed using SDS-PAGE for wild type (A) …
Figure 8 with 1 supplement
Evaluation of expression of Endonuclease G within different mammalian cells and Endonuclease-G-mediated cleavage at mtDNA following shRNA mediated knockdown within cells.
(A) Localization of Endonuclease G in mitochondria in different cell lines. Representative images of localization of Endonuclease G to mitochondria in HeLa, MEF and HEK293T cells. FITC-conjugated …
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Figure 8—source data 1
Localization of Endonuclease G to mitochondria.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig8-data1-v3.zip
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Figure 8—source data 2
Immunofluorescence showing the Localization of Endonuclease G to mitochondria.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig8-data2-v3.zip
Figure 8—figure supplement 1
Analysis of Endonuclease G localization in mitochondria.
Related to Figure 8.
Representative image showing colocalization of Endonuclease G using MitoTracker Green FM (Mt Green) in HeLa cells. Alexa-568 conjugated secondary antibody was used for the detection of Endonuclease G. DAPI is used to stain the nucleus.
Figure 9 with 2 supplements
Investigation of binding efficacy of Endonuclease G to G4 DNA at Region I of mitochondrial genome.
(A) Representative image showing colocalization of Endonuclease G with BG4 in HeLa cells. Alexa Fluor 568 and Alexa Fluor 488 conjugated secondary antibodies were used for detection of Endonuclease …
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Figure 9—source data 1
ChIP assay showing the binding of Endonuclease G with the mitochondrial G-quadruplex regions within cells.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig9-data1-v3.zip
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Figure 9—source data 2
ChIP assay showing the binding of Endonuclease G with the mitochondrial G-quadruplex regions when purified Endonuclease G was used.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig9-data2-v3.zip
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Figure 9—source data 3
P1 nuclease assay showing the binding of Endonuclease G to mitochondrial G quadruples regions.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig9-data3-v3.zip
Figure 9—figure supplement 1
Binding of Endonuclease G to G-quadruplex regions of the mitochondrial genome.
Related to Figure 9. (A) Representative immunofluorescence images showing the colocalization of Endonuclease G and BG4. The ‘Merged’ image shown in left is a colocalization of DAPI, Endonuclease G …
Figure 9—figure supplement 2
Binding of Endonuclease G to G-quadruplex regions of the mitochondrial genome.
Related to Figure 9. (A) SDS profile and western blotting of the pulldown sample when mitochondrial DNA was incubated with mitochondrial extracts. As described in the methodology, mitochondrial …
Figure 10
Investigation of stress conditions that favor the transport of Endonuclease G to mitochondrial matrix.
(A) Western blot showing the presence of Endonuclease G, Cytochrome C and TFAM in either total or supernatant (Sup) and pellet fraction with or without menadione treatment (25 µM) following …
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Figure 10—source data 1
Sub localization of Endonuclease G with or without induction of stress.
- https://cdn.elifesciences.org/articles/69916/elife-69916-fig10-data1-v3.zip
Figure 11
Model depicting mechanism of generation of ‘9 bp deletion’ seen in the mitochondrial genome.
When mitochondria are under stress, Endonuclease G releases into matrix from inner membrane space. In the matrix, Endonuclease G binds and induces cleavage at single-double stranded junctions of G4 …
Tables
Table 1
Oligomers used in the study.
| Oligomer Name | Sequence | Region |
|---|---|---|
| VKK11 | 5’- GCTGTGTCGACTACTACGGTCAATGCTCTG –3’ | GR1 |
| VKK12 | 5’- CTGAGGTCGACTGGGTGATGAGGAATAGTG - 3’ | |
| RBK46 | 5’- TAATCAACACCCTCCTAGCC –3’ | GR2 |
| VKK14 | 5’- GATAGTGTCGACGGCTCATGGTAGGGGTAA –3’ | |
| SD10 | 5'- TTCGCTGACGCCATAAAACT –3' | GR3 |
| SD11 | 5'- ATCAGGGCGTAGTTTGA –3' | |
| SD12 | 5'- GCTCACAAGAACTGCTAA –3' | GR4 |
| SD13 | 5'- TGGATGCGACAATGGAT –3' | |
| SD14 | 5'- TCTTGCACGAAACGGGAT –3' | GR5 |
| SD15 | 5'- TAGGATGAGGATGGATAGT –3' | |
| RBK15 | 5’- CTACTCCTGCTCGCATCTGC –3’ | CR2 |
| RBK16 | 5’- GAAGGTGGTGTTGAGGTTGC –3’ | |
| RBK17 | 5’- GCATTGTTCGTTACATGGTCC –3’ | CR3 |
| RBK18 | 5’- GTGGAAGCGGATGAGTAAGAAG –3’ | |
| RBK19 | 5’- CTCACCACTACAATCTTCCTAG –3’ | CR4 |
| RBK20 | 5’- CAAAGATGGTAGAGTAGATGACG –3’ | |
| RBK21 | 5’- CTAACCATCTTCTCCTTACACCTAG –3’ | CR5 |
| RBK22 | 5’- GTTTGCTAATACAATGCCAGTCAGG –3’ | |
| RBK23 | 5’- CGAAGGTGGATTTAGCAGTAAACTG –3’ | CR6 |
| RBK24 | 5’- CGGTACTATATCTATTGCGCCAGG –3’ | |
| RBK41 | 5’- GTATCATCAACTGATGAGCAAG –3’ | CR1 |
| SD2 | 5’- TCAGCAAACCCTGATGAA –3’ | CR7 |
| SD3 | 5'- CACTCTACTCTCAGTTTACT –3' | |
| SD4 | 5'- ACATCGAATACGCCGCA –3' | CR8 |
| SD5 | 5'- AGTTGGTCGTAGCGGAATCG –3' | |
| SD6 | 5'- TAGGGTTTATCGTGTGAG –3' | CR9 |
| SD7 | 5'- AGTGTGGCGAGTCAGCT –3' | |
| SD8 | 5'- TACTCACTCTCACTGCCCAA –3' | CR10 |
| SD9 | 5'- TGTTTGTCGTAGGCAGAT-3' | |
| VKK21 | 5'- GGATCCATGCGGGCGCTGCGG –3' | |
| VKK22 | 5'- GCGGCCGCTCACTTACTGCCCG –3' | |
| DI12 | 5'- GCAAACCACAGTTTCATGCCCATC –3' | |
| DI13 | 5'- GCCTATAATCACTGCGCCCGCTC –3' | |
| VKK1 | 5’- CCCGTATTTACCCTATAGCACCCCCTCTACCCCC –3’ | C1 |
| VKK2 | 5’- GGGGGTAGAGGGGGTGCTATAGGGTAAATACGGG –3’ | G1 |
| VKK5 | 5’- GTCAGTAGAGGGGGTGCTATAGGGTAAATACGGG –3’ | M1 |
| VKK6 | 5’- GTCAGTAGAGAATGTGCTATAGGGTAAATACGGG –3’ | M2 |
| VKK7 | 5’- GTCAGTAGAGGGGGTGCTATATCATAAATACGGG –3’ | M3 |
| SD 54 | 5'- GGCCAGGGCCCCGCGGTCGAAGCCACTGCC-3' | |
| SD 57 | 5'- TCACCTGGCCGCCGCCGCCAACCAC-3' | |
| DK27 | 5’-TGGGCTCTAGAGGACATAGAGTAAGTGCT-3’ | |
| DK28 | 5’-AGCACTTACTCTATGTCCTCTAGAGCCCA-3’ | |
| KD14 | 5’-CAAGCTCGAAATTAACCCTCAC-3’ | |
| KD13 | 5’-CCCAGTCACGACGTTGTAAAAC-3’ | |
| DI8 | 5’-CTTACAGTGGGCTCTAGAGGGGGTAGATAATAT GCTATAGGGTAAATACTCACTAAAAATCTTTGAA-ATAGGG –3’ | |
| DI9 | 5’-CTAAAAATCTTTGAAATAGGGTGAGTATTTA CCCTATAGCATATTATCTACCCCCTCTAGAGCCCA-CTGTAAG –3’ |
