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

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.Rad27/FEN1核酸酶（Rad27/FEN1 nuclease）在细胞核DNA稳定性维持中发挥关键作用，近期研究发现其可定位于线粒体。已有研究证实，Rad27缺陷会导致线粒体诱变水平升高，但同时会降低线粒体的微卫星不稳定性与同源重组水平。我们针对红霉素耐药相关突变的最新分析显示，其中仅部分突变产生于线粒体DNA（mitochondrial DNA），且GC→AT转换是Rad27纯合缺失背景下线粒体诱变的标志性特征。我们还证实，Rad27缺陷引发的线粒体突变体表型完全依赖于DNA损伤检查点激酶Dun1（DNA damage checkpoint kinase Dun1）。Dun1的失活可抑制Rad27缺陷诱导的线粒体突变体表型，而这一抑制效应可通过后续敲除编码核糖核苷酸还原酶抑制因子的SML1基因得到至少部分解除。我们的研究结论为，Rad27缺陷通过激活DNA损伤检查点激酶Dun1诱发线粒体突变体表型，且Dun1介导的脱氧核苷三磷酸（dNTP）池扩增参与了这一过程。上述结果表明，细胞核DNA不稳定是线粒体诱变的诱因来源。与此一致的是，我们发现缺失Rrm3，一种参与rDNA复制的DNA解旋酶的酵母中更易出现的线粒体突变，同样依赖于Dun1的功能。此外，我们证实，过表达Exo1可抑制线粒体诱变——该蛋白能够缓解Rad27缺陷细胞的细胞核DNA损伤敏感性与复制应激，但无法进入线粒体。Exo1过表达还可恢复Rad27缺陷细胞线粒体中大部分的等位基因重组与微卫星不稳定性水平。与之相反，在rad27纯合缺失背景下，Exo1过表达并不会影响线粒体直接重复序列介导的缺失事件，这表明该同源重组通路是Rad27在线粒体中发挥功能的直接作用靶点。