van Wee Raman, Asor Roi, Li Yiwen, Drechsel David, Popova Mariia, Litos Gabriele, Davidson Iain F, Peters Jan-Michael, Kukura Philipp
Physical and Theoretical Chemistry, Department of Chemistry, Kavli Institute for Nanoscience Discovery, Dorothy Crowfoot Hodgkin Building, University of Oxford, Oxford OX1 3QU, United Kingdom.
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom.
Proc Natl Acad Sci U S A. 2025 Aug 12;122(32):e2514190122. doi: 10.1073/pnas.2514190122. Epub 2025 Aug 5.
DNA loop extrusion by cohesin has emerged as a critical pathway for chromosome organization. In vitro single-molecule experiments indicate that loop extrusion requires the assembly of a heteropentameric complex consisting of the SMC1/SMC3 heterodimer, STAG1, NIPBL, and the kleisin SCC1. The complexity of the complete extrusion machinery, consisting of multiple subunits, DNA binding sites, and ATPases poses substantial challenges for revealing the underlying biomolecular mechanism. As a result, a number of different models have been proposed, many of which do not agree on key mechanistic aspects, such as the details of DNA loading, holoenzyme assembly, or the consequences of ATP binding and hydrolysis. Here, we use mass photometry to comprehensively quantify all the key biomolecular interactions required for DNA loop extrusion. We find that STAG1 binds tightly to the trimeric complex formed by the SMC1/SMC3 heterodimer and SCC1, and together they weakly, but cooperatively, bind the DNA. Full-length NIPBL tightly binds DNA, acting as a DNA anchor during the mechanochemical loop extrusion cycle. Cohesin mutants incapable of head engagement, and those lacking DNA-binding domains in the ATPase heads show negligible differences in overall DNA-affinity, suggesting a minor role of these features for DNA binding. Instead, we find an ATP-modulated DNA binding site created by the interaction of STAG1 with SMC1/SMC3/SCC1, important for repeated grabbing and release of DNA critical to extrusion. Our results call for a careful reexamination of the proposed mechanisms and set energetic boundaries for future proposals.
黏连蛋白介导的DNA环挤压已成为染色体组织的关键途径。体外单分子实验表明，环挤压需要由SMC1/SMC3异二聚体、STAG1、NIPBL和kleisin SCC1组成的异五聚体复合物组装而成。由多个亚基、DNA结合位点和ATP酶组成的完整挤压机制的复杂性，给揭示潜在的生物分子机制带来了巨大挑战。因此，人们提出了许多不同的模型，其中许多在关键机制方面存在分歧，比如DNA加载的细节、全酶组装，或者ATP结合和水解的后果。在这里，我们使用质量光度法全面量化DNA环挤压所需的所有关键生物分子相互作用。我们发现，STAG1与SMC1/SMC3异二聚体和SCC1形成的三聚体复合物紧密结合，它们共同与DNA弱结合，但具有协同作用。全长NIPBL紧密结合DNA，在机械化学环挤压循环中充当DNA锚定物。无法进行头部结合的黏连蛋白突变体，以及ATP酶头部缺乏DNA结合结构域的突变体，在总体DNA亲和力上显示出可忽略不计的差异，这表明这些特征对DNA结合的作用较小。相反，我们发现由STAG1与SMC1/SMC3/SCC1相互作用产生的一个ATP调节的DNA结合位点，这对于重复抓取和释放对挤压至关重要的DNA很重要。我们的结果呼吁对提出的机制进行仔细重新审视，并为未来的提议设定能量边界。
