Topological in vitro loading of the budding yeast cohesin ring onto DNA.

Masashi Minamino,Frank Uhlmann,Céline Bouchoux,Torahiko L Higashi

doi:10.26508/lsa.201800143

Abstract

The ring-shaped chromosomal cohesin complex holds sister chromatids together by topological embrace, a prerequisite for accurate chromosome segregation. Cohesin plays additional roles in genome organization, transcriptional regulation and DNA repair. The cohesin ring includes an ABC family ATPase, but the molecular mechanism by which the ATPase contributes to cohesin function is not yet understood. Here we have purified budding yeast cohesin, as well as its Scc2-Scc4 cohesin loader complex, and biochemically reconstituted ATP-dependent topological cohesin loading onto DNA. Our results reproduce previous observations obtained using fission yeast cohesin, thereby establishing conserved aspects of cohesin behavior. Unexpectedly, we find that non-hydrolyzable ATP ground state mimetics ADP·BeF2, ADP·BeF3 - and ADP·AlFx, but not a hydrolysis transition state analog ADP·VO4 3-, support cohesin loading. The energy from nucleotide binding is sufficient to drive the DNA entry reaction into the cohesin ring. ATP hydrolysis, thought to be essential for in vivo cohesin loading, must serve a subsequent reaction step. These results provide molecular insight into cohesin function and open new experimental opportunities that the budding yeast model affords.

Highlights

Cohesin, a ring-shaped multisubunit protein assembly conserved from yeast to humans, plays crucial roles in chromosome biology (Nasmyth & Haering, 2009; Peters & Nishiyama, 2012; Uhlmann, 2016)
Addition of Scc2–Scc4 resulted in a substantial increase in the ATP hydrolysis rate (Fig 1B)
This is qualitatively similar to the behavior of fission yeast cohesin and consistent with a recent report on ATP hydrolysis by budding yeast cohesin (Murayama & Uhlmann, 2014; Petela et al, 2018)

Summary

Introduction

A ring-shaped multisubunit protein assembly conserved from yeast to humans, plays crucial roles in chromosome biology (Nasmyth & Haering, 2009; Peters & Nishiyama, 2012; Uhlmann, 2016). The cohesin subunits Smc and Smc are characterized by a long stretch of flexible coiled coil, with an ABC family ATPase head domain at one end and a dimerization interface at the other. Dimerization at this interface, known as the “hinge,” generates V-shaped Smc1-Smc heterodimers. The HEAT repeat subunits Scc and Pds, as well as Wapl, contact Scc and regulate cohesin function and dynamics This ring-shaped cohesin complex assembly topologically embraces DNA to promote sister chromatid cohesion (Haering et al, 2008; Murayama et al, 2018)

Methods

Results

Conclusion