Replisomes restrict SMC translocation in vivo

Structural maintenance of chromosomes (SMC) complexes organize genomes by extruding DNA loops. How collisions between SMC complexes and DNA polymerases are resolved in vivo remains poorly understood. Taking advantage of the ability to load SMC complexes at defined sites in the Bacillus subtilis genome, we investigated this problem. We engineered head-on and head-to-tail collisions between SMC complexes and the replisome, and monitored SMC translocation by time-resolved ChIP-seq and Hi-C and replisome progression by marker frequency analysis. We report that SMC complexes do not affect replication progression. By contrast, the replisome blocks SMC translocation regardless of collision orientation. Combining experimental data with simulations, we determined that SMC is first blocked by the replisome and then released from the chromosome. However, occasionally SMC can bypass the replisome and continue translocating. Our findings establish that the replisome is a barrier to SMC-mediated DNA-loop extrusion, with implications for chromosome organization in all organisms. Overall design: Hi-C, ChIP-seq and whole genome sequencing expriments were performed on wild type and mutant cells of Bacillus subtilis PY79 growing in rich medium.

染色体结构维持复合物（Structural maintenance of chromosomes, SMC）通过挤压DNA环来组织基因组。目前学界对体内SMC复合物与DNA聚合酶之间的碰撞如何被解决仍知之甚少。本研究依托可在枯草芽孢杆菌（Bacillus subtilis）基因组特定位点加载SMC复合物的实验手段，对该问题展开探究。我们构建了SMC复合物与复制体之间的头对头及头对尾碰撞模型，并通过时间分辨ChIP-seq与Hi-C技术监测SMC复合物的移位过程，同时借助标记频率分析追踪复制体的行进动态。本研究发现，SMC复合物不会影响复制进程。与之相反，无论碰撞取向如何，复制体均会阻断SMC复合物的移位过程。结合实验数据与模拟结果，我们证实SMC复合物会先被复制体阻断，随后从染色体上解离释放；但偶尔情况下，SMC复合物可绕过复制体并继续完成移位。本研究结果表明，复制体是SMC介导的DNA环挤压过程的屏障，该发现对所有生物的染色体组织调控均具有重要参考价值。实验设计：本研究在富营养培养基中培养的枯草芽孢杆菌PY79野生型与突变体细胞中，开展了Hi-C、ChIP-seq及全基因组测序实验。