Mammalian mitochondrial extracts possess DNA end-binding activity

Gregory Coffey, Uma Lakshmipathy, Colin R Campbell

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

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 heterodimer 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.

Original languageEnglish (US)
Pages (from-to)3348-3354
Number of pages7
JournalNucleic acids research
Volume27
Issue number16
DOIs
StatePublished - Aug 15 1999

Bibliographical note

Funding Information:
We thank G. Chu and M. Zdzienicka, who generously provided the V79 and XR-V15B cell lines, and Dr Susan Critchlow, Wellcome/CRC Institute, UK, who provided polyclonal Ku70/ 80 antibody. We also thank members of the Campbell laboratory for helpful editorial comments. This work was supported in part by grants from the NIH (R29 CA61906), the American Heart Association (9601390) and the American Cancer Society (DHP-171). U.L. was supported by a fellowship from the American Heart Association.

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