Radiation and ROS-induced strand breaks frequently bear strand-break termini that require processing before ligation can occur, including 3-phosphate and phosphoglycolate and 5-hydroxyl end groups (13). PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H2O2-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria. == INTRODUCTION == Single- and double-strand DNA breaks are induced directly by external and internal genotoxic agents such as ionizing radiation (IR), UV light and reactive oxygen species (ROS), or indirectly as a result of aborted topoisomerase action or during base excision repair (BER). Radiation and ROS-induced strand breaks frequently bear strand-break termini that require processing before ligation can occur, including 3-phosphate and phosphoglycolate and 5-hydroxyl end groups (13). Similarly, trapping of topoisomerase 1 by agents such as camptothecin or the presence of abasic sites or nicks adjacent to the cleavage site, followed by tyrosyl-DNA phosphodiesterase 1 (TDP1)-mediated cleavage of the covalent bond linking the DNA to the topoisomerase, generates single-strand breaks with 3-phosphate and 5-hydroxyl termini (4,5). BER performed by bifunctional DNA glycosylases, such as the Nei family members, NEIL1, NEIL2 and NEIL3, remove damaged bases and then cleave the DNA at the abasic sites through a lyase activity that involves ,-elimination to generate 3-phosphate termini (68). The damaged DNA termini have to be restored to 3-hydroxyl and 5-phosphate functionality prior to the completion of the repair process by DNA polymerases and DNA ligases. Polynucleotide kinase/phosphatase (PNKP) plays a major role in the restoration of correct DNA termini following strand cleavage by IR, ROS or NEIL-dependent BER (3,7,9,10). PNKP contains a forkhead-associated domain, which is a proteinprotein interaction domain required for the Temoporfin association of PNKP with CK2-phosphorylated XRCC1 and XRCC4 (1114), and independent DNA 3-phosphatase and 5-kinase domains (15,16). It has been shown that the DNA 3-phosphatase activity of PNKP takes precedence over its DNA 5-kinase activityin vitro(17). Downregulation of PNKP sensitizes cells to IR and hydrogen peroxide (18,19). In addition to damage to nuclear DNA, mitochondrial DNA (mtDNA) is also subject to DNA damage. MtDNA is a 16.5 kbp circular molecule, encoding 37 genes including 13 proteins, 22 tRNAs and 2 rRNAs. Eukaryotic cells can have more than 100 mitochondria and each mitochondrion may contain 10 mtDNAs. In general, mtDNA constitutes about 1% of the total cellular DNA. ROS produced in relatively large quantities in mitochondria during respiration are the major source of mtDNA Temoporfin lesions (20). Damage to mtDNA, if not repaired, can develop into mutations, and mutations of the mtDNA are associated with different diseases including diabetes (21,22), cancer (23), neurodegenerative disorders (24) and aging (25). The rate of mutations in some regions of mtDNA is 20- to 100-fold higher than the nuclear DNA (26). This could be explained by the lack of protection of mtDNA by chromosomal proteins and the proximity of mtDNA to the inner membrane that contains the Temoporfin electron transport chain, which is a constant source of ROS (27). As in the nucleus, BER is the main DNA repair pathway in mitochondria that deals with ROS-induced DNA lesions (8,27,28). Several DNA glycosylases have been identified in mitochondria including Nth and Nei family members (27). Mitochondria also contain a truncated form of APE-1 that can process abasic sites and DNA ends produced by -elimination by DNA glycosylases/lyases such as NTH1 (29). In mitochondrial BER, replacement of missing nucleotides at damaged sites is performed by DNA polymerase (Pol) instead of DNA polymerase found in the nucleus (30), and ligation of DNA at single strand breaks is mediated by DNA ligase III (31). Recent studies have highlighted the GRS importance of mtDNA ligase III for cell survival (31). Topoisomerase 1 and TDP1.