Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3'-phosphatase in maintenance of mitochondrial genome.
ABSTRACT The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3'-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3'-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.
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ABSTRACT: Poly(ADP-ribosylation) is involved in DNA repair and replication, transcription, and cell death. For a long time, only one poly(ADP-ribosylating) enzyme was known, named ADPRT/PARP (EC 126.96.36.199). The recent discovery of a family of PARPs has provided a high degree of complexity in the field. Moreover, the finding that poly(ADP-ribosylation) is not confined to the nucleus but is also carried out by cytoplasmic enzymes supports the idea that it could regulate proteins localized in different cellular compartments. In this respect, a reappraisal of the literature on mitochondrial poly(ADP-ribosylation) could be useful, as well as a discussion of its relevance regarding the current "hot" view of poly(ADP-ribosylation) as a mediator of cell death.The FASEB Journal 11/2004; 18(13):1487-8. · 5.71 Impact Factor
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ABSTRACT: The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.Journal of Biological Chemistry 09/2008; 283(39):26349-56. · 4.77 Impact Factor
Article: Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells.[show abstract] [hide abstract]
ABSTRACT: Exposure to exogenous and endogenous sources cause oxidative damage to cellular macromolecules, including DNA. This results in gradual accumulation of oxidative DNA base lesions, and in order to maintain genomic stability we must have effective systems to repair this kind of damage. The accumulation of lesions is most dramatic in the mitochondrial DNA, and this may cause dysfunction and loss of cellular energy production. Base excision DNA repair (BER) is the major pathway that removes oxidative DNA base lesions, and while we know much about its mechanism in the nuclear DNA, little is yet known about this pathway in mitochondria. While nuclear BER decreases with age, the mitochondrial DNA repair may increase with age. This increase is not enough to prevent the gradual accumulation of lesions in the mitochondrial DNA with age. Accumulation of DNA lesions with age may be the underlying cause for age-associated diseases including cancer.Free Radical Biology and Medicine 06/2002; 32(9):804-12. · 5.42 Impact Factor