DNA Binding by the Mis4 <sup>scc2</sup> Loader Promotes Topological DNA Entrapment by the Cohesin Ring

Abstract

Cohesin, a critical mediator of genome organization including sister chromatid cohesion, is a ring-shaped multisubunit ATPase that topologically embraces DNA. Its loading and function on chromosomes require the Scc2-Scc4 loader. Using biochemical reconstitution, we show here that the loader is a DNA binding protein that directly promotes cohesin loading onto DNA. Two distinct sites within the Mis4Scc2 subunit were found to cooperatively bind DNA. Mis4Scc2 initially forms a tertiary complex with cohesin on DNA and promotes subsequent topological DNA entrapment by cohesin through its DNA binding activity, a process that requires an additional DNA binding surface provided by Psm3Smc3, the ATPase domain of cohesin. In support, we show that mutations in the two DNA binding sites of Mis4 impair the chromosomal loading of cohesin. These observations demonstrate that physiological importance of DNA binding by the loader and provide mechanistic insight into the process of topological cohesin loading.