Grant: Travel Awards - Institute Community Support

Introduction: The effective transfer of genetic information during cell division requires a major structural reorganization of chromosomes. A nuclear enzyme called condensin triggers this process known as chromosome condensation. This enzyme is a pentameric ATPase complex composed of a catalytic core of two SMC proteins (Smc2-4) and three regulatory subunits. The ATPase domain of condensin is essential to chromatin compaction since abrogation of this activity in Smc2 or Smc4 leads to the inactivation of the complex and lethality. However, how ATP hydrolysis contributes to condensin functions in vivo is still poorly understood. Methods and Results: To explore ATP hydrolysis role in chromosome condensation, we took advantage of the significant similarities shared by SMC ATPase domains and the extensively studied ABC-type transporters. We designed a collection of mutants affecting the Smc4 ATPase domain with the objective of progressively reducing its catalytic activity without completely disabling nucleotide hydrolysis. By using five fold dilution assay, we identified mutants that affect Smc4 functionality to different degrees. Through fluorescence in situ hybridization (FISH), we characterized how these mutations affect chromosome condensation. Among all the mutants studied, mutations in H-loop and C-helix ATPase submotifs specially compromised chromosome compaction. Finally the analyses of condensin ATPase activity on the mutants of interest confirmed that all had less activity than the will type form. All this result together reveals a direct correlation between the severity of the thermosensitive phenotype associated with ATP hydrolysis defects and condensation abnormalities. Conclusion and Relevance: Further studies with these mutants will allow us to unravel the mechanism of chromosome condensation in eukaryotes and, in particular, how condensin harnesses the energy of ATP hydrolysis to compact chromatin in vivo.