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

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）是一类在细胞核DNA稳定性维持中发挥关键作用的酶，近日研究发现其可定位于线粒体。据此，已有研究证实Rad27缺失会导致线粒体中诱变活性增强，但微卫星不稳定性（microsatellite instability）与同源重组（homologous recombination）水平降低。我们针对红霉素抗性突变的当前分析显示，仅部分此类突变产生于线粒体DNA，且GC→AT转换（GC→AT transition）是rad27敲除背景下线粒体诱变的标志性特征。我们还证实，Rad27缺失所引发的线粒体突变表型（mitochondrial mutator phenotype）完全依赖于DNA损伤检查点激酶（DNA damage checkpoint kinase）Dun1。Dun1失活可抑制Rad27缺失引发的线粒体突变表型，而后续敲除编码核糖核苷酸还原酶（ribonucleotide reductase）抑制因子的SML1，可至少部分解除这一抑制效应。我们得出结论：Rad27缺失通过激活DNA损伤检查点激酶Dun1引发线粒体突变表型，且Dun1介导的dNTP库（dNTP pools）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源头。与之相符的是，我们证实，在缺失参与rDNA复制（rDNA replication）的DNA解旋酶（DNA helicase）Rrm3的酵母中，线粒体突变发生频率更高，且此类突变同样依赖于Dun1。此外，我们证实，Exo1过表达可抑制Rad27缺失细胞的细胞核DNA损伤敏感性与复制应激，且该蛋白无法进入线粒体，同时可抑制线粒体诱变。Exo1过表达还可恢复Rad27缺失细胞线粒体中大部分的等位基因重组（allelic recombination）与微卫星不稳定性。与之相反，在rad27敲除背景下，Exo1过表达不会影响线粒体中直接重复序列介导的缺失（direct-repeat mediated deletions），这表明该同源重组通路（homologous recombination pathway）是Rad27在线粒体中发挥活性的直接作用靶点。