RNA-DNA hybrids on protein coding genes are stabilized by loss of RNase H and are associated with DNA damages during S-phase in fission yeast. Schizosaccharomyces pombe 972h-

RNA-DNA hybrid is a part of the R-loop which is an important non-standard nucleic acid structure. RNA-DNA hybrid-R-loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA-DNA hybrids and map their genomic locations in fission yeast cells. The RNA-DNA hybrids appear as multiple nuclear foci in rnh1rnh201 mutant cells lacking cellular RNase H activity, but not in the wild-type. The majority of RNA-DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1rnh201 mutant cells, cells with multiple Rad52 foci increase during S-phase and about 20% of the RNA-DNA hybrids overlap with Rad52 loci. During S-phase, more robust association of Rad52 with RNA-DNA hybrids was observed in the protein coding region than in M-phase. These results suggest that persistent RNA-DNA hybrids in the protein coding region in rnh1rnh201 mutant cells generate DNA damages during S-phase, potentially through collision with DNA replication forks.

RNA-DNA杂交体是R环（R-loop）的组成部分，而R环是一类重要的非标准核酸结构。RNA-DNA杂交体-R环可通过诱导DNA损伤或抑制DNA复制引发基因组不稳定，同时在转录、复制、重组与修复的调控过程中发挥重要的生物学功能。本研究采用催化失活的人源核糖核酸酶H1（RNase H1）突变体（D145N），对裂殖酵母细胞内的RNA-DNA杂交体进行可视化并绘制其全基因组定位图谱。在缺失细胞核糖核酸酶H活性的rnh1rnh201突变体细胞中，RNA-DNA杂交体表现为多个细胞核灶，而野生型细胞中未观察到此现象。绝大多数RNA-DNA杂交位点分布于蛋白质编码区与转运RNA（tRNA）区域。在rnh1rnh201突变体细胞中，S期阶段带有多个Rad52核灶的细胞比例上升，且约20%的RNA-DNA杂交位点与Rad52位点存在重叠。在S期，蛋白质编码区中Rad52与RNA-DNA杂交体的结合强度显著高于M期。上述结果表明，rnh1rnh201突变体细胞中蛋白质编码区的持久性RNA-DNA杂交体可在S期诱发DNA损伤，其潜在机制可能是与DNA复制叉发生碰撞。