Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, we have mapped RNA:DNA hybrids, replication stress markers and DNA double strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids were found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR was only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also cumulated at TTS, leading to persistent checkpoint activation, spreading of ?-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner. Overall design: NGS-based analysis of the effect of R-loops on replication stress and genomic instability in human cells

R环（R-loops）对染色体功能兼具正向与负向调控效应。为鉴定人类基因组中的毒性R环，我们对拓扑异构酶I（Topoisomerase I，Top1）敲低的细胞开展了分析：该酶可松弛DNA超螺旋并抑制R环形成，我们在此类细胞中绘制了RNA:DNA杂交体（RNA:DNA hybrids）、复制应激标志物以及DNA双链断裂（DSBs）的全基因组定位图谱。
RNA:DNA杂交体富集于高表达基因的启动子（转录起始位点，TSS）与终止子（转录终止位点，TTS）区域。与之相反，ATR介导的复制蛋白A（RPA）磷酸化仅在TTS区域被检测到，而TTS区域相较于转录方向呈现优先的头对头复制取向。
在Top1敲低的细胞中，DSBs同样在TTS区域累积，进而引发持续性检验点激活、染色质上?-H2AX的扩散以及全基因组复制叉速度减缓。上述数据表明，在携带R环的高表达基因的TTS区域发生的复制叉停滞，可阻止复制与转录之间的头对头冲突，并以Top1依赖的方式维持基因组完整性。
总体实验设计：基于下一代测序（NGS）的分析，探究人类细胞中R环对复制应激与基因组不稳定性的影响。