Mitochondrial Localization Of XPD Protein And Its Function In Mitochondrial DNA Oxidative Damage Repair

Xeroderma pigmentosum group D (XPD/ERCC2) encodes an ATP-dependent helicase that plays essential roles in both transcription and nucleotide excision repair (NER) of nuclear DNA. In cytoplasm, XPD interacts with MMS19 forming MMXD complex mainly functioning in mitotic spindle formation and chromosome segregation. Oxidative DNA damage induction is demonstrated to be several folds higher in mtDNA relative to nuclear DNA and XPD-deficient cells are hypersensitive to oxidative DNA damages. While the essential role of XPD in maintaining genomic stability has been well defined however, whether or not XPD exerts similar functions in mitochondrial DNA oxidative damage repair remains elusive.In this study, we provide the first evidence that XPD is localized in the inner membrane of mitochondria. Our immunofluorescent staining data showed that XPD was present in both the nuclear, cytoplasmic and mitochondrial compartments, which was further confirmed by Western blotting analysis on fractionated cell lyses showing the presence of the XPD protein in the mitochondrial fraction. Further, proteinase K and alkaline treatments demonstrated that XPD was located in the inner membrane of mitochondria. Our observations of the mitochondrial localization of XPD together with oxidative damage specific enrichment of XPD in mitochondria clearly support the notion that XPD may be a crucial DNA repair factor for oxidative DNA damage in mitochondria. Consistent with its mitochondrial localization, mitochondrial ROS production and levels of oxidative stress-induced mitochondrial DNA (mtDNA) common deletion were significantly elevated, whereas capacity for oxidative mtDNA damage repair and the level of ATP and mitochondrial membrane potential were markedly reduced in both XPD-suppressed human osteosarcoma (U2OS) cells and XPD-deficient human fibroblasts. The fact that XPD deficiency selectively affects the oxidative DNA damage repair capacity of mtDNA but not nuclear genes suggests that XPD is a crucial factor for oxidative DNA damage repair machinery operating in mitochondria.The potential interacting factors with XPD in the repair of mitochondrial oxidative DNA damages were then explored by immunoprecipitation together with mass spectrometric analysis on mitochondrial fraction, and two novel physical proteins including TUFM and MMS19 were detected which were further validated by Co-IP analysis. TUFM is a mitochondrial Tu translation elongation factor and MMS19 can form a complex with XPD in cytoplasm and nucleus. However, there is no interaction between TUFM and MMS19. Similar to the findings in XPD-deficient cells, a markedly increased level of mitochondrial common deletion and a significantly decreased capacity for repairing mitochondrial oxidative DNA damages were observed after TUFM or MMS19 expression in mitochondria was suppressed. Thus, similar to its interacting partner XPD, TUFM and MMS19 also play a role in oxidative mtDNA damage repair.Our findings clearly demonstrate that XPD plays crucial role(s) in protecting mitochondrial genome stability by facilitating an efficient repair of oxidative DNA damage in mitochondria. XPD forms two kinds of complexes XPD-TUFM and XPD-MMS19, which are critical for an efficient formation of oxidative DNA damage repair machinery in mitochondria.