Genetic Evidence that the Acetylation of the Smc3p Subunit of Cohesin Modulates Its ATP-Bound State to Promote Cohesion Establishment in Saccharomyces cerevisiae

Abstract

Sister chromatid cohesion refers to the process by which sister chromatids are tethered together until the metaphase-to-anaphase transition. The evolutionarily conserved cohesin complex mediates sister chromatid cohesion. Cohesin not only ensures proper chromosome segregation, but also promotes highfidelity DNA repair and transcriptional regulation. Two subunits of cohesin (Smc1p, Smc3p) are members of the structural maintenance of chromosomes (SMC) family. The SMC family is recognized by their large coiled-coil arms and conserved ATP-binding cassette-like ATPase domain. While both Smc1p and Smc3p ATP binding and hydrolysis are essential for cohesin function in vivo, little is known about how this core enzymatic activity is regulated to facilitate sister chromatid cohesion. Here we use SMC mutant proteins to block specific steps in cohesin's ATPase cycle in Saccharomyces cerevisiae. We show that blocking Smc3p-mediated ATP binding or Smc3p ATP hydrolysis traps unique functional states in cohesion. Finally, we provide evidence that Smc3p acetylation, which has an essential role in cohesion establishment, modulates the Smc3p ATP-bound state.

In vitro assembly of physiological cohesin/DNA complexes

“…To test whether our in vitro system also mimicked this dependency, we reduced cohesin affinity to ATP with a K38I mutation in the ATP-binding cassette of Smc3p. This mutation inhibits both ATP binding to Smc3p and in vivo cohesin binding to chromosomes (6,8). Yet, it does not affect the integrity of the complex (6,8).…”

“…Another important in vivo parameter of cohesin binding to chromosomes is its dependency on binding and hydrolysis of ATP by Smc1p and Smc3p (6)(7)(8). To test whether our in vitro system also mimicked this dependency, we reduced cohesin affinity to ATP with a K38I mutation in the ATP-binding cassette of Smc3p.…”

“…This mutation inhibits both ATP binding to Smc3p and in vivo cohesin binding to chromosomes (6,8). Yet, it does not affect the integrity of the complex (6,8). We prepared S-phase extracts from cells expressing Smc3p and either wild-type Smc3p-6HA or smc3p-6HA K38I, and assayed C1-C substrate binding via Western blot using HA antibodies.…”

“…In vivo assembled cohesin/chromosome complexes can be very stable as evidenced by the inability to elute cohesin from isolated chromosomes with 1.6 M salt (5). Binding to chromosomes also requires ATP binding to both Smc1p and Smc3p (6)(7)(8). In all organisms, cohesins bind to specific regions on chromosome arms as well as to large domains in the pericentric region (9,10).…”

Cohesin Acetylation Promotes Sister Chromatid Cohesion Only in Association with the Replication Machinery

“…Genetically, the effect of Smc3 acetylation is to counteract cohesion disruption by the Wapl/Rad61 protein (1,3,17), although other models have been proposed (43). Genetic analysis also points to a functional interaction between Smc3 acetylation and the nucleotide cycle of the cohesin complex (44). Thus Wapl, whose activity is suppressed by cohesin acetylation, may work by stimulating cycles of ATP exchange or hydrolysis by Smc3, causing cohesin to unload from chromatin.…”