Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae

Abstract

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.

The peroxiredoxin Tsa1 extends the lifespan of budding yeast by maintaining the stability of the ribosomal RNA gene cluster

“…RAD27 encodes a structure-specific nuclease that plays important roles in maintaining the integrity of nuclear genomes. Rad27 is also localized in the mitochondria and is involved in maintaining mitochondrial DNA stability (Kaniak-Golik et al, 2017). TSA1 encodes a Prx that scavenges H 2 O 2 , which suppresses the accumulation of mutations (Huang et al ., 2003).…”

The GTPase Arf1 Is a Determinant of Yeast Vps13 Localization to the Golgi Apparatus

“…To construct strains for the mtDNA escape assay, a mtDNA donor kar1-1 strain was constructed to facilitate the transfer of mtDNA from the PTY44 strain [81] to MATa rho 0 strains. To do so, the strain JC8/55 [82] was crossed to PTY44 [81] as described [83]. Mating mixtures were diluted 1000× and plated on SC medium supplemented with leucine (at 20 mg L −1 ) and canavanine (at 20 mg L −1 ).…”