Enhanced expression of the DNA damage-inducible gene DIN7 results in increased mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae

Abstract

We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.

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

“…It has been shown that overexpression of Din7 destabilizes poly(GT) in mtDNA and enhances gross rearrangements in mtDNA, presumably due to enhanced recombination (129). The Sia group has shown that loss of the Din7 function does not significantly affect repeat-mediated mtDNA deletions, but it synergizes with the G776D allele of MSH1 (see below) to increase this particular recombination-based process (130).…”

Yeast Exonuclease 5 Is Essential for Mitochondrial Genome Maintenance

“…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).…”

“…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).…”

Analysis of Repeat-Mediated Deletions in the Mitochondrial Genome of Saccharomyces cerevisiae

“…The DIN7 gene codes for a mitochondrial protein homologous to the S. cerevisiae Exo1p, Rad2p, and the flap endonuclease Rad27p (Mieczkowski et al 1997;Fikus et al 2000). Overexpression of Din7p results in a twofold increase in recombination between mutations at the cytb gene that are 1 kb apart (Koprowski et al 2003). Although these proteins have biochemical activities consistent with a role in recombination, their loss has not been demonstrated to result in a substantial defect in this process in vivo.…”