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.
Highlights
The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration
The resulting gap filling by a DNA polymerase and nick-sealing by DNA ligase during base excision repair (BER) could proceed via two subpathways: single nucleotide (SN)-BER where only the damage base is replaced or LP-BER where 2– 6 additional nucleotides at the 5Ј terminus are removed by flap endonuclease 1 (FEN-1) followed by resynthesis
Purity of Mitochondrial Extracts—It is evident that DNA repair proteins are not exclusively localized in the nucleus or mitochondria, and that a variable but significant fraction could be found in the cytoplasm
Summary
The absence of an aldehyde group in the oxidized deoxyribose fragment at the 5Ј terminus after DNA strand break precludes removal of these lesions by the dRP lyase activity of pol  in the nucleus In such a case the 5Ј-blocking group is removed by flap endonuclease 1 (FEN-1), a 5Ј-exo/endonuclease [17, 18]. In contrast to the situation in the nucleus with multiple enzymes, pol ␥ and lig3␣ are involved in both replication and BER of mtDNA [19] In this case, 5Ј-dRP generated after AP site cleavage is removed by the dRP lyase activity of pol ␥, similar to that of pol  for the nuclear SN-BER. We provide evidence for LP-BER in human and mouse mitochondria mediated primarily by an unidentified 5Ј-exo/endonuclease
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