Repair of DNA double-strand breaks in mitochondria: Implications in mitochondrial genome maintenance

Abstract

Mitochondrial DNA (mtDNA) is frequently exposed to oxidative damage compared to nuclear DNA. mtDNA deletions are associated with mitochondrial disorders. Deletions identified in humans are flanked by short direct repeats; however, mechanism of DNA rearrangements is yet to be elucidated. Besides maintenance of genomic stability in mitochondria is poorly understood.  Here, we investigate the mechanisms of DSB repair in mitochondria. While classical-NHEJ was undetectable, microhomology mediated endjoining (MMEJ) efficiently repaired DSBs in mitochondria. Immunoblotting, immunoprecipitation and other assays suggest the involvement of CtIP, FEN1, MRE11 and PARP1 in mitochondrial MMEJ. Knockdown experiments demonstrated that DNA LIGASE III, but not LIGASE IV or LIGASE I, is primarily responsible for final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ and its functions in deletions and in many human mitochondrial disorders. Further, we show that HR mediated repair is more efficient in mitochondria of testes as compared to brain, kidney and spleen. Interestingly, sequence analyses revealed a predominant reciprocal exchange mechanism, while 35% consisted of gene conversion. Colocalization and immunoblotting studies revealed the role of MRN complex in HR mediated repair in mitochondria. These observations highlight the importance of HR in mitochondrial genome maintenance.