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The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence supports the conclusion that the nuclear DNA ligase III protein plays an essential role in both base excision repair and homologous recombination. However, the role of DNA ligase III protein in mitochondrial genome dynamics has been obscure. Human tumor-derived HT1080 cells were transfected with an antisense DNA ligase III expression vector and clones with diminished levels of DNA ligase III activity identified. Mitochondrial protein extracts prepared from these clones had decreased levels of DNA ligase III relative to extracts from cells transfected with a control vector. Analysis of these clones revealed that the DNA ligase III antisense mRNA-expressing cells had reduced mtDNA content compared to control cells. In addition, the residual mtDNA present in these cells had numerous single-strand nicks that were not detected in mtDNA from control cells. Cells expressing antisense ligase III also had diminished capacity to restore their mtDNA to pre-irradiation levels following exposure to gamma-irradiation. An antisense-mediated reduction in cellular DNA ligase IV had no effect on the copy number or integrity of mtDNA. This observation, coupled with other evidence, suggests that DNA ligase IV is not present in the mitochondria and does not play a role in maintaining mtDNA integrity. We conclude that DNA ligase III is essential for the proper maintenance of mtDNA in cultured mammalian somatic cells.

Hamster EM9 cells, which lack Xrcc1 protein, have reduced levels of DNA ligase III and are defective in nuclear base excision repair. The Xrcc1 protein stabilizes DNA ligase III and may even play a direct role in catalyzing base excision repair. Since DNA ligase III is also thought to function in mitochondrial base excision repair, it seemed likely that mitochondrial DNA ligase III function would also be dependent upon Xrcc1. However, several lines of evidence indicate that this is not the case. First, western blot analysis failed to detect Xrcc1 protein in mitochondrial extracts. Second, DNA ligase III levels present in mitochondrial protein extracts from EM9 cells were indistinguishable from those seen in similar extracts from wild-type (AA8) cells. Third, the mitochondrial DNA content of both cell lines was identical. Fourth, EM9 cells displayed no defect in their ability to repair spontaneous mitochondrial DNA damage. Fifth, while EM9 cells were far more sensitive to the cytotoxic effects of ionizing radiation due to a defect in nuclear DNA repair, there was no apparent difference in the ability of EM9 and AA8 cells to restore their mitochondrial DNA to pre-irradiation levels. Thus, mitochondrial DNA ligase III function is independent of the Xrcc1 protein.

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.

DNA end-joining was measured by incubating linearized plasmid DNA with mitochondrial protein extracts. A spectrum of end-joined molecules ranging from re-circularized monomer to dimer and higher molecular weight forms was observed. The DNA end-joining reaction required ATP and Mg2+, and was inhibited by sodium chloride. Both cohesive- and blunt-ended DNA molecules were end-joined, although the former were more efficient substrates. Molecular analysis of rejoined molecules revealed that >95% of the linearized DNA were precisely end-joined. The few imprecisely end-joined molecules recovered, sustained deletions that spanned direct repeat sequences. The deletions observed are strikingly similar to those present in mitochondrial genomes of patients with Kearns-Sayre or Pearson syndromes, certain ophthalmic myopathies and the aged. These results suggest that mammalian mitochondria possess a DNA double strand break repair activity similar to that seen in the nucleus, and that this repair pathway may play a role in the generation of mitochondrial DNA deletions associated with a number of human pathologies.