Supplementary MATERIALS
Supplementary Figure Legends
Supplementary Figure 1. Comparison of HR efficiency in mitochondrial extract at varying reaction conditions. A. Bar diagram showing the mean fold change of recombination frequency when increasing concentrations of mitochondrial extracts (1, 2 and 3 μg) were used. B. Bar diagram showing the mean fold change in recombination frequency of mitochondrial extracts at different incubation timepoints (0, 15, 30, 60, 120 min). C. Table shows HR efficiency in the absence of either dNTPs, ATP or NAD in the buffer. Testicular mitochondrial extract with the buffer containing all the components served as a positive control, while the reaction without protein served as the negative control. Data shown is derived from three independent repeats.D. Table showing the significance values when experimental samples were compared with control. ANOVA was performed using Tukey’s multiple comparison test for statistical analysis. Significance values presented are *p<0.05.
Supplementary Figure 2. HR efficiency when transformed into RecA+ bacterial strain. A. Table showing the recombination frequency when Tg1 bacterial cells were transformed with recombination products obtained after incubation in mitochondrial extracts. Tg1 is a RecA+ E. coli strain and has its own recombination mechanism. B. Bar diagram showing the difference in recombination activity when DNA from no protein control (NPC) was transformed into DH5α (RecA-) and Tg1 (RecA+). Standard deviation was calculated and shown for cumulative of three biological repeats (ns: not significant, *p< 0.05, **p< 0.005, ***p< 0.0001).
Supplementary Figure 3. Evaluation of HR efficiency of whole cell extracts among different rat tissues. A. Table showing the recombination frequency when different whole cell extracts were used for HR assay. B. Bar diagram comparing the mean fold change of recombination frequency of different tissues with respect to control.
Supplementary Figure 4. Efficiency of HR mediated DSB repair in mitochondrial extracts prepared from HeLa cells. A. Table showing the recombination frequency of increasing concentration of mitochondrial extracts prepared from HeLa cells. Mitochondrial extracts from rat testicular cells were used as positive control. B. Immunoblot analyses showing purity of HeLa cell mitochondrial extracts. PCNA is used as nuclear and cytosolic marker, while CYTOCHROME C served as a mitochondrial marker. ACTIN served as a loading control.
Supplementary Figure 5. DNA sequencing of recombinants resulting due to reciprocal exchange and gene conversion. A. Chromatogram showing the full sequence of a recombinant resulting from reciprocal exchange. Sequences within the red oval depict the EcoRI and SalI sites. Red dotted box depicts the sequence restoration at neomycin gene. B. Chromatogram showing the full sequence of a recombinant resulting from gene conversion. Sequences within the oval depict the EcoRI, HindIII and SalI sites. Red dotted box depicts the sequence of neomycin gene restored following recombination. C. The relevant portions of chromatograms showing all the recombinant clones resulting from reciprocal exchange with characteristic restriction enzyme sites represented by red oval. D. Relevant portion of chromatograms showing all the recombinant clones resulting from gene conversion with characteristic restriction enzyme sites represented by red oval.
Supplementary Figure 6. Localization of RAD50, RAD51, MRE11, NIBRIN and Ligases in mitochondria as assessed by immunofluorescence. Representative images of colocalization of RAD50, RAD51, MRE11, NIBRIN, LIGASE III, LIGASE I and LIGASE IV with mitochondria in HeLa cells. LIGASE IV expression was mostly restricted to nucleus and was undetectable in mitochondria, which was consistent to previous reports. FITC conjugated secondary antibodies were used for detecting proteins. Mt-DR is Mitotracker Deep Red, a mitochondria specific dye. DAPI was used for nuclear stain. For LIGASE IV, nuclear stain was given as pseudo color. B. Colocalization analyses using JaCoP in ImageJ software. Minimum of 50 cells were used for analysis of colocalization of red and green signals, and plotted as the colocalization value of green overlapping red (dot plot marked as green). Y-axis depicts the Mander’s colocalization coefficient value calculated for green over red and plotted in the form of dot plot. C. Colocalization analyses values plotted as red overlapping green (dot plot marked as red). Y-axis depicts the Mander’s colocalization coefficient value calculated for red over green and plotted in the form of dot plot. The significance for both the plots was calculated using GraphPad Prism 5.0 with respect to secondary control.
Supplementary Figure 7. Schematic showing proposed mechanism of genome stability in mitochondria. Schematic presentation showing different modes of DSB repair in mitochondria. When DSBs are introduced in mitochondrial DNA, repair can take place through homologous recombination (using a homologous copy of the DNA) or if the break is flanked by direct repeats, using microhomology mediated end joining. HR, is error free and can be mediated either through reciprocal exchange or gene conversion, thus helping in maintaining the genomic stability. MMEJ is error prone and results in deletions of varying lengths.
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