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. 2016 Jan 15;27(2):223-35.
doi: 10.1091/mbc.E15-05-0260. Epub 2015 Nov 25.

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Satish Kumar Tadi  1 , Robin Sebastian  1 , Sumedha Dahal  1 , Ravi K Babu  1 , Bibha Choudhary  2 , Sathees C Raghavan  3
Affiliations

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Satish Kumar Tadi et al. Mol Biol Cell. .

Abstract

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

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Figures

FIGURE 1:
FIGURE 1:
Comparison of efficiency of classical NHEJ in cell-free extracts and in mitochondrial extracts prepared from various rat tissues. (A, B) Evaluation of purity of mitochondrial extracts. Mitochondrial and cell-free extracts were prepared from rat tissues (brain, testes, spleen, kidney) and analyzed for specific markers by Western blotting. CE, cytoplasmic extract; ME, mitochondrial extract. CEs and MEs were probed with VDAC and cytochrome C (mitochondrial markers), with β-actin as loading control (A). PCNA and β-actin (nuclear and CFE markers, respectively) were used as the markers to test the purity of fractions. (C) Schematic representation of the oligomeric substrates (5′ compatible, noncompatible [5′-5 and 5′-3′], and blunt ends) mimicking endogenous DSBs used for the study. (D–G) Bar diagrams showing quantification of the end-joined products from mitochondrial extracts from the brain (D), testes (E), spleen (F), and kidney (G) using 5′-compatible, noncompatible, and blunt ends. WCE extracts from respective rat tissues were used as positive control. For NHEJ assay, 5-μg extracts were incubated with [γ-32P]ATP-labeled oligomeric DNA substrate for 2 h at 30°C (testis) or 37°C. The reaction products were deproteinized and resolved on 8% denaturing PAGE. For quantification, the highest photostimulated light unit (PSLU) value of end joining was taken as 100%, and the relative efficiency between different extracts was calculated. Experiments were repeated a minimum of three times.
FIGURE 2:
FIGURE 2:
Evaluation of microhomology-mediated DNA end joining in MEs prepared from rat tissues. (A) Schematic presentation of the microhomology (13 nt) containing substrate used and expected MMEJ reaction product (63 base pairs) after PCR amplification. Region of microhomology is indicated in blue and red in the substrates. (B) Denaturing PAGE profile showing MMEJ observed after PCR amplification catalyzed by MEs from rat brain, testes, spleen, and kidney. Cell-free extracts from respective tissues served as the control. The arrow indicates expected 63-nt-long dimeric product resulting from MMEJ. Other bands above the MMEJ product are due to microhomology-independent NHEJ products. M, 60-nt marker. For MMEJ assay, 5-μg extracts were incubated with oligomeric ds DNA substrates for 2 h at 25°C, and the end-joined products were PCR amplified using [γ-32P]ATP- labeled primers and resolved on 8% denaturing PAGE. (C) Bar diagram showing quantification of the end-joined products based on multiple experiments. The efficiency of the joining was calculated. For quantification, the highest PSLU value of C-NHEJ or MMEJ was taken as 100%, and the relative efficiency within different extracts was calculated. (D) XcmI digestion of MMEJ products of mitochondrial extracts from the brain, testes, spleen, and kidney. XcmI digestion depends on recreation of the microhomology region upon joining, as indicated in A. MMEJ products were incubated with XcmI (5 U/10 μl reaction) at 37°C for 2 h, and the products were resolved on 8% denaturing PAGE. (E) Cloning and sequencing of MMEJ junctions derived from mitochondria of testes, spleen, and kidney. The nucleotides in blue represent the retained microhomology region, and red shows deletion. Green represents the junction between random sequence and the start of microhomology. Pink indicates mutations. All NHEJ junctions shown are from different clones and are derived from independent PCR and transformations. (F) Mechanism of MMEJ based on sequence analysis.
FIGURE 3:
FIGURE 3:
Evaluation of length and position of microhomology on efficiency of MMEJ in mitochondria. (A) Gel profile showing efficiency of MMEJ in mitochondrial extracts when length of microhomology is 3, 5, 8, or 10 nt. (B) Bar diagram showing quantification of MMEJ and C-NHEJ products shown in A. Graph is a cumulative representation of three independent repeats. For quantification, highest PSLU value of C-NHEJ or MMEJ was taken as 100%, and the relative efficiency between different extracts or substrates was calculated. (C) Denaturing PAGE profile showing efficiency of MMEJ in mitochondrial extracts when the length of the microhomology is 13, 16, 19, or 22 nt. (D) Bar graphs representing quantification of the C-NHEJ and MMEJ products shown in C. In all cases, cell-free extracts from testes served as the control. For quantification, the highest PSLU value of C-NHEJ or MMEJ was taken as 100%, and the relative efficiency between different extracts or substrates was calculated. (E) Comparison of MMEJ efficiency of mitochondrial extracts when an 8-nt microhomology region was positioned at different lengths from a DSB. Here, 8+8 indicates that the distance between the microhomology and DSB is 8 nt in both substrates; 24+24, that the distance between microhomology and DSB is 24 nt in each substrate; and while 8+24 and 24+8 that the distance is either 8 or 24 nt as indicated. DNA substrates were designed in such a way that microhomology- mediated joining creates a restriction site for XmnI. The band at 50 nt is contributed by random primer extension of unused DNA substrates where radiolabeled primer can bind. (F) Bar diagram showing quantification of the end-joined products of the gels shown in E. In all cases, MMEJ products are indicated by arrows, and C-NHEJ products are bracketed. For quantification, the highest PSLU value of C-NHEJ or MMEJ was taken as 100%, and the relative efficiency between different extracts or substrates was calculated. M′, γ-32P–labeled 50-nt ladder; M, labeled 60-nt oligomer.
FIGURE 4:
FIGURE 4:
Immunoblot analyses showing the presence and level of expression of DSB repair proteins in mitochondria of various rat tissues. (A) Comparison of level of expression of C-NHEJ proteins in mitochondrial extracts and CFE prepared from rat tissues. The ∼30-μg extracts from the brain, testes, spleen, and kidney were resolved on 8–10% SDS–PAGE, transferred to polyvinylidene fluoride membrane, and probed using the respective antibodies. (B) Presence and expression of MMEJ and other DSB repair proteins in mitochondrial extracts of various tissues and corresponding whole-cell extracts.
FIGURE 5:
FIGURE 5:
Efficiency of mitochondrial MMEJ after immunodepletion and inhibitor-based inactivation of C-NHEJ and alternate NHEJ proteins. (A) Bar diagrams showing the quantification of efficiency of immunodepletion in mitochondrial extracts. β-Actin was used as loading control. (B) Evaluation of efficiency of MMEJ in mitochondrial extracts after immunodepletion assayed using 13-nt microhomology substrates. (C) Bar diagram showing quantitation of MMEJ efficiency in immunodepleted rat tissue mitochondrial extracts based on multiple gels. For quantification, the relative intensity of the MMEJ band between different samples was calculated and expressed as PSLU. (D–I) Comparison of the effect of repair inhibitors against ATM kinase (KU55933), DNA-PK (NU7026), ligase I (L82), PI3K (wortmannin), MRE11 (mirin), and PARP1 (3-ABA) on MMEJ of 13-nt microhomology substrates. For quantification, the relative intensity of the MMEJ band between different samples was calculated and is expressed as PSLU (bar graphs). We preincubated 1- to 2-μg rat tissue mitochondrial extracts with inhibitors with increasing concentration (10 μM to 10 mM) for 30 min on ice and subjected them to MMEJ assay for 2 h at 25°C. The end-joined products were PCR amplified using γ-32P–labeled primers and resolved on 8% denaturing PAGE. In the case of control reactions, for which inhibitor was not added, and equal concentration of DMSO was used (see Supplemental Figure S7 for details). *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 6:
FIGURE 6:
Mitochondrial DNA ligase III, but not ligase IV, is required for mitochondrial EJ inside the cells. (A) Immunoblot analyses showing the purity of cytosolic extracts (CEs) and mitochondrial protein extracts (MEs) prepared from HeLa cells transfected with antisense DNA ligase III plasmid. We used ∼15-μg extracts from HeLa cells (CE, ME, and CFE) for immunoblotting. Cytochrome C, PCNA, and β-actin were used as mitochondrial, nuclear, and CFE markers, respectively. (B) Immunoblot analysis showing knockdown of DNA ligase III from HeLa cells and its quantification. For quantification, expression in MOCK ME was taken as 100, and relative expression in AS L3 ME was calculated. β-Actin was used as a loading control. (C) MMEJ assay using HeLa mitochondrial extracts prepared from mock and antisense DNA ligase III–transfected cells on 13-nt microhomology–containing DNA substrates. Bar diagram showing relative reduction in MMEJ efficiency in antisense DNA ligase III–transfected cells compared with the mock transfection. (D) Immunoblot analysis showing knockdown of DNA ligase IV in HeLa cells transfected with mock and antisense DNA ligase IV plasmid. Tubulin was used as internal loading control. (E) Comparison of MMEJ in mitochondrial extracts after knockdown of ligase IV in HeLa. MMEJ activity in CFE served as the control. (F) Bar diagram showing relative reduction in C-NHEJ but not in MMEJ efficiency in antisense DNA ligase IV–transfected cells compared with the control transfection shown in D. *p < 0.05, **p < 0.01, ***p < 0.001.
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