RESUMO
The cohesin ring, which is composed of the Smc1, Smc3, and Scc1 subunits, topologically embraces two sister chromatids from S phase until anaphase to ensure their precise segregation to the daughter cells. The opening of the ring is required for its loading on the chromosomes and unloading by the action of Wpl1 protein. Both loading and unloading are dependent on ATP hydrolysis by the Smc1 and Smc3 "head" domains, which engage to form two composite ATPase sites. Based on the available structures, we modeled the Saccharomyces cerevisiae Smc1/Smc3 head heterodimer and discovered that the Smc1/Smc3 interfaces at the two ATPase sites differ in the extent of protein contacts and stability after ATP hydrolysis. We identified smc1 and smc3 mutations that disrupt one of the interfaces and block the Wpl1-mediated release of cohesin from DNA. Thus, we provide structural insights into how the cohesin heads engage with each other.
Assuntos
Acetiltransferases/genética , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Dimerização , Hidrólise , Modelos Moleculares , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , CoesinasRESUMO
Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes.