**4.3 Mismatch repair (MMR)**

MMR is a highly conserved pathway involved in the correction of misincorporation and slippage mistakes committed by polymerases during DNA replication, and base mismatches generated by base deamination, alkylation, and oxidation [3]. In general terms, MMR process presents the next steps: localization of the mismatch and identification of the newly synthesized strand, excision at both extremes of the mismatch lesion, DNA resynthesis, and finally ligation to complete the process [10].

 The first demonstration of mammalian mitochondria capable to repair mismatch lesions was done by Mason et al. [51]. They observed that rat liver mitochondrial lysates repaired G-T and G-G mismatches; however, by using immunodetection no MSH2, a key nuclear element in MMR, was detected in these lysates, suggesting that mitochondrial MMR activity uses different elements to those of the nucleus. Posteriorly, in the pursuit of proteins responsible for the activity observed, one study reported that mismatches and small IDLs in mitochondrial genome are recognized by the Y-box binding protein 1 (YB1), which also localizes to nucleus where it exerts other functions, and its depletion in cultured cells triggers an increased mutagenesis in mtDNA. In addition, it was demonstrated that MMR activity is independent of MSH2 and that MSH3, MSH6 or MLH1 are not present in human mitochondria, at least under the experimental conditions employed [52]. In contrast to the previous observation, it was later demonstrated that MLH1 do localizes to mitochondria [53]. Through overexpression of *Mlh1* or *Msh2* in retinal endothelial cells, it was determined that MLH1 has a protective role in mtDNA after glucose-induced DNA damage, and on the other hand, this protective effect was not detected when *Msh2* was overexpressed, observation that suggests no participation in mtDNA maintenance [54], in accordance with previous studies. Additionally, it was reported that the incidence of base-mismatches in mtDNA in diabetic retina is a consequence of expression silencing of MLH1 by methylation of its promoter, activity performed by Dnmt1, enzyme overexpressed in diabetes. Thus, these observations propose that MLH1 has an important role in mtDNA maintenance, since its silencing by methylation triggers mtDNA damage [55]. In summary, MMR pathway is involved in mitochondrial genome maintenance; however, not all the elements implicated have been found. It could be possible that the proteins of mitochondrial MMR may have a different splicing or post translational versions than nuclear ones, which impairs their identification through antibody-based techniques, or in the other hand, mitochondrial MMR could not depend of all the elements involved in the canonical nuclear form, as it was seen with the participation of YB1 [52]. In any case, more research is needed to find more MMR nuclear factors within mitochondria or to discover new ones and be able to catalog the mitochondrial MMR as an original pathway.

### **4.4 Single strand break repair (SSBR)**

The repair of SSBs in mitochondria is achieved through a BER subpathway known as base excision/SSB repair (which is also present in nucleus), since both mechanisms share common component, especially in the last steps: gap filling and DNA ligation [10, 24]. Indeed, most of the SSBs can be repaired by elements of BER pathway: APE1, PKPK, and Polγ [24]. Other members of mitochondrial SSB repair include: PARP1, a protein implicated in the detection of SSBs in the nucleus and also more recently observed in mitochondria, where not only it binds to mtDNA, but also when is depleted, this provokes accumulations of DNA damage, thus confirming its participation in mtDNA maintenance [56]. Besides, the participation of ExoG in SSB repair has been elucidated, since its depletion induces mitochondrial persisting SSBs that eventually lead to apoptosis [57].

 As was previously mentioned, there are some lesions associated to SSB, like trapped topoisomerase 1 (Top1), damage that can be repaired through the action of tyrosil-DNA phosphodiesterase 1 (TDP1), an important enzyme involved in the release of covalently trapped Top1 with DNA that was first described in yeast [58]. In addition to its well characterized function, it has been observed that TDP1 also removes other types of 3′-blocking lesions, resulting oxidative damage [59–61]. A fraction of TDP1 (nuclear encoded) localizes to the mitochondria, where it has been implicated in mtDNA repair, since the treatment with chain terminator nucleotide analogs (CTNAs), which are also substrates of this enzyme, in *tdp1*<sup>−</sup>/<sup>−</sup> cells generate a reduction in mtDNA copy number, whereas wild type cells remain unaffected [61, 62]. These findings confirm the involvement of TDP1 in mtDNA damage repair, in this case induced by CTNAs. Another SSB-related lesion is the generation of a covalent binding of adenine monophosphate (AMP) to the 5′end of mtDNA, and this error is promoted by abortive ligase activity. The resolution of such damage relies in aprataxin (APTX) protein that is able to remove 5′-adenylate groups. APTX localizes to mitochondria, whereas its depletion generates a decline of mtDNA copy number as well as higher levels of DNA damage, observations that suggest a direct role of this enzyme in mtDNA maintenance [63]. If any of the lesions mentioned remains unrepaired, further complications may appear, since SSBs may progress to DSBs, which are more deleterious to cells.

#### **4.5 Double strand break repair (DSBR)**

In general, cells of higher eukaryotes use two main approaches to repair DSBs. The first approach is through the union of the ends in a nonhomologous dependent way, this pathway is termed nonhomologous end joining (NHEJ); it has been determined that NHEJ possesses some alternatives versions that use noncanonical elements, these sub pathways are known as alternative NHEJ (A-NHEJ); the repair with these mechanisms guarantee the restoration of DNA integrity but not sequence.

#### *4.5.1 Nonhomologous end joining (NHEJ)*

Nonhomologous end joining (NHEJ) is one of the two main pathways used by the cells to repair DNA double strand breaks. Similar to most DNA repair processes, NHEJ is based on three general steps: the action of a nuclease to resect the damaged DNA, next, the fill-in to make new DNA by a polymerase, and finally the participation of a ligase to restore the integrity of the strands. One of the most interesting features of NHEJ is the diversity of substrates that can use and convert to joined products [64]. By virtue of its template-independent operation, NHEJ is associated with insertions and deletions and hence with a lack of reliable restoration [26].

It has been observed that mammalian mitochondria do possess the capacity to bind DNA ends, activity that is retained even in Ku-deficient cells [65], additionally, the efficiency and precision of this activity apparently depend on the structure of the ends generated, since blunt-ended DNA fragment repaired are less conserved than sticky ends in comparison to the original [66]. Tadi et al. [67] demonstrated that mitochondria have a noncanonical version of NHEJ, also named alternative NHEJ (A-NHEJ). The repair by this pathway is based on microhomology and is sometimes associated with long deletions, and hence it is described as microhomology-mediated end joining (MMEJ). In this same study, using rodents and human mitochondrial extracts, a lack of end-to-end joining of nonligatable broken DNA was observed, the fact that suggests the absence of a functionally operative canonical NHEJ in mitochondria, or at least is undetected with the techniques used. In contrast, mitochondria have the ability to join oligomeric dsDNAs

#### *Mitochondrial Genome Maintenance: Damage and Repair Pathways DOI: http://dx.doi.org/10.5772/intechopen.84627*

harboring direct repeats (microhomology) that vary in length, from 5 to 22 nt, with an efficiency that is enhanced with the increase in the length of homology. These results are supported by a previous observation, where DSBs are induced in mice through mitochondrially targeted restriction endonuclease (*Pst* I), and the repaired mtDNA presented small directed repeats (a few nucleotides) at the breakpoint [68], resolution that fits with the repair manner of MMEJ, and in addition, these repair products have also been observed in most of the mtDNA deletions associated with human diseases, which are mostly (~85%) flanked by small direct repeats [69]. Besides, it has been determined that this mitochondrial MMEJ activity involved the proteins CtIP, FEN1, Mre11, PARP1, and ligase III [67].

Therefore, MMEJ has been proposed as a central pathway in the repair of double strand breaks and maintenance of mammalian mitochondrial genome, and the use of this pathway and possibly not C-NHEJ may explain the Ku independence proteins to exert the joining activity as was previously mentioned. The possible absence of C-NHEJ activity in mitochondria is contrasting with the observation that antibodies to KU70 and KU80 cross-react with proteins from mitochondrial extracts with DNA end-binding activity [10, 70]. Furthermore, it has been determined that XRCC4, a mediator protein of nuclear DSB repair pathway, is present in mitochondrial, where it is indeed involved in mtDNA repair and possibly associated with DNA ligase III [48]. In summary, at present no NHEJ activity has been described in mitochondria; however, this organelle presents the ability to join broken DNA ends, and it appears that this action depends on the structure of the ends generated and in the presence of homology at both ends of the DSB, further, the repair by MMEJ may explain the deletions observed in the majority of mitochondrial diseases.

### *4.5.2 Homologous recombination (HR)*

Homologous recombination (HR) is a ubiquitous process conserved from bacteriophages to humans and is one of the most important pathways used by the cells to deal with DNA double strand breaks. To achieve the restoration of molecular integrity and sequence in a free-error manner, HR needs a homologous sequence to use it as a template [26, 71].

 It has been determined that HR is essential for preservation of mtDNA in plants, yeast, and fungi, and on the other hand, although there is evidence about HR in mammalian, its significance in vivo is not clear [1]. One of the first reports about mitochondrial homologous repair capabilities was made by Thyagarajan et al. [72] where they observed that mitochondrial protein extracts from mammalian cells catalyzed homologous recombination repair of plasmid DNA substrates, therefore concluding that mitochondria do possess the machinery to perform this process. Additionally, after preincubating protein extracts with anti-RecA antibodies, an inhibition of the reaction was observed, fact that suggests the participation of a mammalian mitochondrial RecA homolog. Supporting this evidence, in 2010, Sage et al. [73] demonstrated that Rad51 and the related proteins, Rad51C and XRCC3, localize to human mitochondria, and they also reported that the protein levels were enriched after stress induction and that depletion of any of these elements generates a dramatic decrease in mtDNA copy number, these results strongly suggest some type of HR participation in mitochondrial genome maintenance. Other proteins involved in HR have been observed in mitochondria, and their participation in mtDNA repair has been validated, such as Dna2 [74] and Mre11 [67, 75, 76]; moreover, it has been suggested that ExoG could supply Exo1, and therefore, many of the factors needed to perform HR process are present in mitochondria.

Recently, using biochemical assays, it was determined that HR is the major DSBR mechanism, where it has a role in maintenance of mitochondrial genome

 integrity, since the induction of DSBs significantly enhanced this process. Besides the participation of Rad51, Mre11 and Nibrin relevance in HR was confirmed by suppression of HR-mediated repair after immunodepletion of these proteins in the mitochondrial extracts [76]. The process of mitochondrial HR may proceed in two ways, one through intramolecular recombinant events, where a sole mtDNA molecule recombines with itself, and a second form, where a molecule can recombine with another one homologous or heterologous [77, 78]. Despite the knowledge of the elements involved in mitochondrial HR have increased over the last years, the exact mechanism about how HR is achieved in mitochondria is lacking in comparison to the nuclear models [33].

 The second approach is through the use of nondamaged homologous sequences; this kind of repair restores molecular integrity as well as sequence; another pathway that uses homology sequences is single strand annealing (SSA), which needs directed repeats in both ends of the DSB, in such a way that when repairing, it restores integrity and sequence but at the expense of a variable length deletion [26].
