Abstract：
A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.
摘要：
DNA双链断裂(DSB)危害基因组完整性并危及细胞活力。主动转录的基因如果被破坏并且需要被抑制则是特别有害的。然而,抑制启动的速度有多快，以及它对染色体上相邻基因的影响有多大，仍然难以捉摸。我们采用了最近开发的，非常快速的CRISPR以精确的时间在特定的基因组位点产生DSB，可视化活细胞中的转录，并测量断裂位点附近的RNA聚合酶II(RNAPII)占有率。我们观察到单个DSB在几分钟内抑制受损基因的转录，这与损伤修复蛋白的募集相吻合。转录抑制从DSB沿着染色体双向传播数百千碱基，在这个过程中，蛋白酶体被诱发以去除RNAPII。我们的方法为测量单个DSB周围的快速动力学事件和阐明分子机制奠定了基础。
