TY - JOUR
T1 - Topological in vitro loading of the budding yeast cohesin ring onto DNA
AU - Minamino, Masashi
AU - Higashi, Torahiko L.
AU - Bouchoux, Céline
AU - Uhlmann, Frank
N1 - Funding Information:
The authors thank N Patel and A Alidoust from the Crick Fermentation Science Technology Platform for their support and members of our laboratory for discussions and critical reading of the manuscript. This work was supported by an EMBO Long Term Fellowship to M Minamino, the European Research Council (grant agreement no. 670412), and by The Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001198), the UK Medical Research Council (FC001198), and the Wellcome Trust (FC001198).
Publisher Copyright:
© 2018 Minamino et al.
PY - 2018
Y1 - 2018
N2 - 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. In this study, 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 nonhydrolyzable 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, believed to be essential for in vivo cohesin loading, must serve a subsequent reaction step. These results provide molecular insights into cohesin function and open new experimental opportunities that the budding yeast model affords.
AB - 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. In this study, 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 nonhydrolyzable 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, believed to be essential for in vivo cohesin loading, must serve a subsequent reaction step. These results provide molecular insights into cohesin function and open new experimental opportunities that the budding yeast model affords.
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U2 - 10.26508/lsa.201800143
DO - 10.26508/lsa.201800143
M3 - Article
C2 - 30381802
AN - SCOPUS:85057040991
VL - 1
JO - Life Science Alliance
JF - Life Science Alliance
SN - 2575-1077
IS - 5
M1 - e201800143
ER -