Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils [Hi-C]. Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils [Hi-C]

The Structural Maintenance of Chromosome (SMC) protein complexes cohesin, condensin and the Smc5/6 complex (Smc5/6) are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6’s recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 controls the spatial organization of supercoiled chromosomal regions. The results show that Smc5/6 associates with transcription-induced positively supercoiled chromosomal DNA at cohesin-dependent chromosome loop boundaries. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes, and efficiently initiates loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA. Overall design: Analysis of chromosome structure by Hi-C in various mutants in Saccharomyces cerevisiae

染色体结构维持（Structural Maintenance of Chromosome, SMC）蛋白复合物——黏连蛋白复合体（cohesin）、凝聚蛋白复合体（condensin）与Smc5/6复合物（Smc5/6）——对染色体功能至关重要。在分子层面，这类复合物通过环挤出（loop extrusion）方式折叠DNA。据此，黏连蛋白复合体可在细胞间期形成染色体环，而凝聚蛋白复合体则能压缩有丝分裂期的染色体。然而，Smc5/6新近发现的DNA环挤出活性所承担的生物学功能尚不明确。本研究揭示，Smc5/6可调控超螺旋染色体区域的空间构象。实验结果表明，Smc5/6可结合于依赖黏连蛋白复合体的染色体环边界处、由转录诱导产生的正超螺旋染色体DNA上。从机制层面分析，单分子成像实验显示，Smc5/6二聚体可特异性识别正超螺旋DNA缠结环（plectoneme）的顶端，并高效启动环挤出过程，将超螺旋DNA汇聚为大型缠结环。最后，高通量染色体构象捕获（Hi-C）分析显示，Smc5/6可在顺式作用层面连接包含转录诱导正超螺旋的染色体区域。综上，本研究结果表明，Smc5/6可通过识别正超螺旋DNA并启动环挤出过程，调控染色体的三维空间构象。实验整体设计：在酿酒酵母（Saccharomyces cerevisiae）的各类突变体中，通过高通量染色体构象捕获（Hi-C）分析染色体结构。