Ribodysgenesis: sudden genome instability in the yeast Saccharomyces cerevisiae arising from RNase H2 cleavage at genomic-embedded ribonucleotides

Yeast Saccharomyces cerevisiae has been widely used as a model system for studying genome instability. Here, heterozygous S. cerevisiae zygotes were generated to determine the genomic alterations induced by sudden introduction of active RNase H2. In combination of a custom SNP microarray, the patterns of chromosomal instability could be explored at a whole genome level. Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term “ribodysgenesis”. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.酿酒酵母（Saccharomyces cerevisiae）已被广泛用作研究基因组不稳定性的模式实验系统。本研究通过构建杂合酿酒酵母合子，以探究主动型核糖核酸酶H2（RNase H2）瞬时导入所诱导的基因组改变。结合定制化单核苷酸多态性微阵列（SNP microarray）技术，可在全基因组层面解析染色体不稳定性的特征模式。 核糖核苷酸可在DNA复制过程中由复制性DNA聚合酶嵌入基因组DNA。这类异常DNA亚基可被高效识别，并通过由异源三聚体酶核糖核酸酶H2（RNase H2）启动的核糖核苷酸切除修复（Ribonucleotide Excision Repair）通路予以清除。在高等真核生物中，核糖核酸酶H2是必需基因；而酿酒酵母即便缺失核糖核酸酶H2也可存活，尽管其基因组的突变、重组及其他基因组不稳定性事件的发生率会显著升高。 尽管核糖核酸酶H2可被视为基因组的保护因子，能够抵御因识别并移除嵌入基因组的核糖核苷酸所引发的有害事件，但在核糖核酸酶H2缺陷型菌株中，若基因组内存在大量嵌入且滞留的核糖核苷酸时，主动导入核糖核酸酶H2会引发大规模DNA断裂与基因组不稳定性，我们将这一状态命名为“核糖核苷酸基因组紊乱（ribodysgenesis）”。 此类DNA断裂与基因组不稳定性仅由靶向含核糖核苷酸基因组的核糖核酸酶H2切割所介导。核糖核苷酸基因组紊乱后的存活菌株会出现大量杂合性缺失（loss of heterozygosity）事件，此类事件源于含核糖核苷酸DNA上的重组损伤，其发生率较野生型菌株提升超过1000倍。DNA断裂现象会在1至2次细胞分裂周期内产生，随后细胞会适应基因组中存在的核糖核酸酶H2与核糖核苷酸，恢复至正常基因组不稳定性水平并正常增殖。