Molecular Mechanisms of Mitochondrial Genetic Activity

When isolated, respiring mitochondria of Saccharomyces cerevisiae are exposed to ethidium bromide (EtdBr) they are capable of catalyzing the following discrete reactions. mtDNA + mEtdBr (Me2+)/→ [EtdBrm·DNA'] (1) [Etdbrm·DNA'] + (ATP) (nuclease(s))→ fragments + ... (2) (Reactions 1 + 2) mtDNA (3) + mtEtdBr + (ATP) → fragments + ... nATP ([EtdBrm·DNA'])/→ nADP + nPi (4) In this scheme (EtdBrm·DNA'] represents a novel, stable derivative of EtdBr, linked (probably covalently) to mitochondrial DNA fragments (DNA') which, as isolated, have a mean mass of 12.5 x 106 daltons. This intermediate had previously been shown to be formed in vivo and in vitro with kinetics of appearance and degradation consistent with it being the first product in the mutagenic sequence known to be initiated by EtdBr. Experiments using respiratory inhibitors and uncouplers suggest that Reaction 1 depends on an energized state of the inner membrane, while Reactions 2 and 3 are driven by ATP, either added externally or generated by oxidative phosphorylation. Simultaneously there is an enhancement of the hydrolysis of added ATP to ADP plus Pi (Reaction 4). Experiments with inhibitors, plus the use of mutants with lesions either in their mitochondrial ATPase complex or in enzyme(s) involved in the repair and recombination of their DNA, have shown that Reactions 2 (and hence 3) and 4 are tightly linked. Thus the DNAse (Reaction 3) depends on an intact coupling device and the EtdBr-induced uncoupling (Reaction 4) is dependent on the presence of mtDNA and appropriate enzyme(s) for its degradation. The addition of uncouplers, such as chlorocarbonylphenylhydrazone or colicin K, disrupts this link by providing an alternate pathway for the activation of ATPase and leads to an immediate cessation of Reaction 2. Euflavine, a known antagonist to EtdBr in vivo has no effect on any of the reactions by itself, but blocks Reactions 2, 3, and 4 when added simultaneously with or subsequent to EtdBr. Strains of S. cerevisiae known to be resistant to mutagenesis by EtdBr all exhibit a lowered rate of Reaction 3 but for different reasons: some exhibit a lowered capacity to form [EtdBr·DNA'], others to bring about its degradation. Obligately aerobic, petite negative yeasts are known not to be affected by EtdBr in a permanent fashion: mitochondria of all these strains show a complete inability to catalyze Reaction 1.