In the baker's yeast, Saccharomyces cerevisiae, more than half of all apparently nonspecific nuclease activity is located within the mitochondrion. We have purified that activity and have shown that the enzyme is composed of a single polypeptide with a molecular weight of 38,000 daltons. It is nuclear encoded, bound to the mitochondrial inner membrane, and digests single-stranded RNA and DNA as well as double-stranded DNA; the latter substrate it degrades both endo- and exonucleolytically (5' to 3'). What function does this highly active, broad specificity nuclease have within the mitochondrion? In yeast, mitochondrial gene expression requires in a highly specific manner the processing of long, polycistronic primary transcripts into the mature monocistronic RNAs. Furthermore, mitochondrial DNA recombination is so highly proficient that pairs of genetic markers separate by as little as 50-100 base pairs can show 1% recombination in crosses. We propose to test the hypothesis that the major mitochondrial nuclease is required for one or both of those processes. We have raised antibodies to the purified enzyme and will use these as a probe to isolate its gene sequence from a library of genomic DNA in the lambdalgtll expression vector. The cloned gene sequences will be employed to construct mutants by gene disruption methods in which the chromosomal gene will be inactivated. Such null mutants, as well as thermosensitive classes we will isolate, will be characterized in order to assess the role of the nuclease in mitochondrial RNA processing and DNA recombination. In the long range, these mutants will be an invaluable resource for studying the biochemistry of those events, including RNA splicing, in mitochondrial extracts. Such studies are less feasible now because of the overwhelming nucleolytic activity in extracts due to their """"""""contamination"""""""" with the major mitochondrial nuclease. The diversity of post-transcriptional processing events observed with mitochondrial RNAs is an indication of their importance for gene expression. Characterizing the enzymes and mechanisms involved in RNA processing at a biochemical level is a necessary step towards understanding mitochondrial gene regulation and the nuclear-cytoplasmic interactions that control mitochondrial biogenesis in yeast.
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