Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.
Graduate Program in Cellular and Molecular Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.
Mol Cell Biol. 2023;43(6):254-268. doi: 10.1080/10985549.2023.2199660. Epub 2023 May 13.
Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops and self-associating domains. Purified cohesin diffuses along DNA in an ATP-independent manner but can be propelled by transcribing RNA polymerase. In conjunction with a cofactor, the complex also extrudes DNA loops in an ATP-dependent manner. In this study we examine transcription-driven translocation of cohesin under various conditions in yeast. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by an inducible gene. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers capture mobilized cohesin demonstrates that transcription-driven complexes translocate processively in vivo. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.
黏合蛋白是染色体的核心结构元件，调节着许多基于 DNA 的事件。该复合物将姐妹染色单体保持在一起，直到有丝分裂前期开始，并将各个染色体 DNA 组织成环和自聚集结构域。纯化的黏合蛋白在无 ATP 的情况下沿 DNA 扩散，但可以被转录 RNA 聚合酶推动。在与辅助因子结合的情况下，该复合物还可以以 ATP 依赖的方式挤出 DNA 环。在这项研究中，我们研究了酵母中各种条件下转录驱动的黏合蛋白易位。为此，将越来越大的障碍物系在 DNA 上，作为受诱导基因驱动的复合物的障碍。障碍物由 GFP-lacI 核心融合到一个或多个 mCherries 构建而成。一个带有四个 mCherries 的嵌合体在 G1 晚期阻止了黏合蛋白的通过。在 M 期，阈值障碍取决于凝聚状态：非凝聚复合物也被四个 mCherries 阻止，而凝聚复合物则被三个 mCherries 阻止。此外，在障碍物处停滞的凝聚复合物反过来也阻止了非凝聚复合物的通过。合成障碍捕获了被动员的黏合蛋白，证明了转录驱动的复合物在体内进行连续易位。总的来说，这项研究揭示了黏合蛋白在染色体上运动的未被探索的限制。
