Yeast Exonuclease 5 Is Essential for Mitochondrial Genome Maintenance

Abstract

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 exo5⌬ 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 RNAto-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 5Ј 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 5Ј 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 ␥ 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 ...

Identification of a novel human mitochondrial endo-/exonuclease Ddk1/c20orf72 necessary for maintenance of proper 7S DNA levels

“…Comparison of reaction products with a reference RNA fragment of the same length as the one present in the chimeric RNA–DNA substrate, as well as to an RNA ladder, showed that the major final product contained two deoxyribonucleotides downstream of the RNA fragment (Figure 5B). A similar feature was observed for another PD-(D/E)XK nuclease EXO5 (48). The second substrate (DNA–RNA) seemed fully degraded because the DNA portion of the molecule that was labelled can be hydrolyzed; however, as Ddk1 has no RNase activity, we assume that the RNA portion remains, although not visible in the autoradiogram (Figure 5B).…”

“…An ssDNA substrate was treated with Ddk1, and the migration of reaction products was compared with a DNA ladder. This revealed that the major final product is 2 nucleotides in length, which is the same as observed for yeast EXO5 (48) (Figure 6B).…”

“…The yeast genome encodes a mitochondrialy localized nuclease DEM1/EXO5 that, as Ddk1, belongs to the PD-(D/E)XK superfamily, and although a highly distant relative in terms of sequence, exhibits similar catalytic properties to Ddk1 (36,48). EXO5, which lacks RNase activity, can degrade a RNA–DNA chimeric substrate by sliding across RNA and cleaving within the DNA.…”

“…EXO5, which lacks RNase activity, can degrade a RNA–DNA chimeric substrate by sliding across RNA and cleaving within the DNA. Like Ddk1, EXO5 does not digest circular substrates, but in contrast to Ddk1, it acts from the 5′ to 3′ end (48). The role of EXO5 has not been fully elucidated but has been shown to be essential for mitochondrial genome maintenance, as its inactivation generates petites with unstable mitochondrial genomes that continuously undergo rearrangements (48).…”

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

“…A 5= DNA exonuclease activity, defined by the EXO5 gene, has been recently identified in yeast mitochondria (132,133). EXO5 is essential for the maintenance of ϩ but not the ori-containing hypersuppressive Ϫ genomes.…”