Yeast Exonuclease 5 Is Essential for Mitochondrial Genome Maintenance

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Δ strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5′ exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5′ double-stranded DNA or 5′ RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.Endonucleases and exonucleases are intimately involved in all aspects of DNA metabolism in the cell. In mitochondria, several constitutive nucleases have been identified that contribute to the proper maintenance of the mitochondrial genome through replication and recombination pathways. In addition, nucleases can localize to mitochondria in response to DNA stress in order to mediate appropriate DNA repair. Among the constitutive mitochondrial nucleases in Saccharomyces cerevisiae are the Nuc1 nuclease that contributes to DNA recombination efficiency and functions in apoptosis (4, 38) and the Cce1 endonuclease that resolves recombination intermediates (29). The Din7 endonuclease is a mitochondrially located 5′ flap endonuclease related to FEN1 (20). While deletion of the gene for either of these enzymes produced marginal mitochondrial phenotypes, more severe phenotypes were observed when combined deletions of these nuclease genes were studied or when they were combined with deletions of other genes involved in DNA recombination or repair, such as MHR1 or MSH1 (20, 22, 27). Recently, human Dna2 was shown to localize to both the nuclear and mitochondrial compartments and to participate in mitochondrial DNA replication and base excision repair (11, 39). Its function in yeast mitochondrial DNA maintenance has not been studied in detail. Finally, the 5′ flap endonuclease FEN1, which normally functions in primer RNA degradation during Okazaki fragment maturation in the nucleus, also localizes to the mitochondrion in response to DNA damage, participating in long-patch base excision repair (19, 23).Since mitochondrial function is not essential to yeast survival, dysfunction caused by mutations of the mitochondrial genome can be readily detected as a loss of respiration function, which is scored as the inability to grow on nonfermentable carbon sources. A defect in the mitochondrial DNA polymerase γ MIP1 results in complete loss of the mitochondrial DNA, and the mutant fails to grow on glycerol-containing media lacking glucose (14). Such cells are designated ρ⁰. Genome maintenance defects can also result in the generation of petite mutants that still contain mitochondrial DNA. Generally, most of the mitochondrial genome has been deleted, and a small origin-containing region has been amplified (ρ⁻). S. cerevisiae contains eight such origin regions that are highly similar in sequence and are distributed over the 86-kb mitochondrial genome (8, 9, 15). Petites that have amplified the ori5 region have been studied more extensively (16, 22).While the nucleases listed above participate in the proper maintenance of the mitochondrial genome through their replication and/or recombination functions, none appears to be essential for the integrity of the mitochondrial genome. One reasonable explanation for these observations is functional redundancy. Indeed, functional nuclease redundancy is quite common; it has been observed in the process of DNA degradation during mismatch repair in Escherichia coli, during Okazaki fragment maturation in yeast, and during the resection of double-stranded breaks in yeast (7, 25, 33). However, the possibility remains that an additional nuclease(s) is active in the mitochondrion. The present paper describes an essential mitochondrial exonuclease that is distantly related to the nuclease domain of RecB, a subunit of the bacterial RecBCD recombinase. This nuclease was discovered over 2 decades ago during a biochemical chromatographic survey of yeast exonucleases and was called exonuclease 5 (3). Initial studies with a partially purified enzyme preparation showed it to be a 5′ exonuclease specific for single-stranded DNA (ssDNA). Here we report the identification of the EXO5 gene and describe comprehensive biochemical and genetic studies that show a critical role for EXO5 in mitochondrial DNA maintenance, presumably through the processing of replication intermediates. Upon deletion of EXO5 or inactivation of its nuclease activity, only ρ⁻ mutants could be recovered. EXO5 has previously been characterized as DEM1 (defects in morphology) because the deletion mutant shows defects in growth and in mitochondrial morphology (10, 12). No nuclear defect associated with an EXO5 deletion has been detected.