Cohesin, a critical mediator of genome organization including sister chromatid 
cohesion, is a ring-shaped multi-subunit ATPase that topologically embraces DNA. 
Its loading and function on chromosomes require the Scc2-Scc4 loader. Using 
biochemical reconstitution, we show here that the ability of the loader to bind 
DNA plays a critical role in promoting cohesin loading. Two distinct sites 
within the Mis4Scc2 subunit are 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. Furthermore, we show that mutations in the two DNA 
binding sites of Mis4 impair the chromosomal loading of cohesin. These 
observations demonstrate the physiological importance of DNA binding by the 
loader and provide mechanistic insights into the process of topological cohesin 
loading.