Abstract

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains (TADs) and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy (HS-AFM) imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1 and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, HS-AFM imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA binding mechanism mediated by dynamic protein conformational changes.