Mammalian mitochondrial extracts possess DNA end-binding activity

Abstract

Mammalian mitochondrial protein extracts possess DNA end-binding (DEB) activity. Protein binding to a 394 bp double-stranded DNA molecule was measured using an electrophoretic mobility shift assay. Mitochondrial DEB activity was highly specific for linear DNA. Inclusion of a vast excess of non-radioactive circular DNA did not disrupt binding to radioactive f394. In contrast, binding was abolished by the inclusion of linear competitor DNA. In mammals, nuclear DEB activity is due to Ku, a hetero-dimer composed of the Ku70 and Ku86 proteins. To determine whether mitochondrial DEB activity was also due to Ku, protein extracts were prepared from the Chinese hamster XR-V15B cell line, which lacks this protein. As anticipated, nuclear extracts prepared from these cells lacked DEB activity. In contrast, mitochondrial extracts prepared from these cells had wild-type levels of DEB activity, demonstrating that this latter activity is not a consequence of nuclear contamination. Although the nuclear and mitochondrial DEB activities are independent of each other, they are nevertheless closely related, since mitochondrial DEB activity was 'supershifted' by both anti-Ku70 and anti-Ku86 antisera. The nuclear DEB protein Ku plays an essential role in nuclear DNA double-strand break repair. The DEB activity described herein may therefore play a similar role in mitochondrial DNA repair.

Mitochondrial Genome Maintenance: Roles for Nuclear Nonhomologous End-Joining Proteins inSaccharomyces cerevisiae

“…However, more direct experimental evidence for recombination and end joining in mammalian mitochondria, derived from in vivo and in vitro studies, has accumulated in recent years (Poulton et al 1993;Thyagarajan et al 1996;Coffey et al 1999;Lakshmipathy and Campell 1999;Kajander et al 2001;Kraytsberg et al 2004). The finding that some of the known nuclear DSBR proteins are localized to the mitochondria has suggested that, like BER, some of these pathways may be operational in both nuclear and mitochondrial compartments.…”

Mre11 is expressed in mammalian mitochondria where it binds to mitochondrial DNA

“…Recombinational repair mechanisms apparently exist since in vitro studies demonstrated homologous recombination and end-joining activities in mammalian mitochondrial extracts (6,25,41). When DSBs are induced in mitochondria by restriction endonucleases, both intramolecular and intermolecular recombination products with large deletions appear (2,19).…”

Using Genetic Reporters to Assess Stability and Mutation of the Yeast Mitochondrial Genome

“…Other DNA repair proteins have been shown to affect mitochondrial DNA maintenance in mammalian cells, including the BER flap endonuclease FEN1 (Liu et al, 2008), DNA doublestrand break repair proteins, Rad51p (Sage et al, 2010), Mre11 (Dmitrieva et al, 2011) and Ku80 (Coffey et al, 1999), and the nucleotide excision repair protein CSA (Kamenisch et al, 2010). In addition, in yeast, DNA damage tolerance pathways that utilize the translesion polymerase complexes encoded by Rev1p, Rev3p, and Rev7p also impact mitochondrial mutagenesis (Kalifa and Sia, 2007;Zhang et al, 2006).…”