Mammalian mitochondrial extracts possess DNA end-binding activity.
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.
Coffey
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G
Department of Pharmacology, University of Minnesota Medical School, 3-249 Millard Hall, 435 Delaware Street SE, Minneapolis, MN 55455, USA.
Lakshmipathy
U
U
Campbell
C
C
D000818:Animals; D034961:Antigens, Nuclear; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D008928:Mitochondria / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen