Biochemical studies on the pre-replication complex of Archaea
Several proteins play a crucial role in the formation of the pre-replication complex (pre-RC) in eukaryotes: origin recognition complex (ORC), CDC6 and minichromosome maintenance proteins (MCM). In yeast, these proteins associate with DNA in a sequence-specific manner during the cell cycle. Archaea, the third kingdom of life, are believed to replicate DNA in a eukaryote-like fashion. Most archaeal species encode single MCM and two CDC6/ORC proteins. The reduced number of replication proteins suggests that archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication. This thesis describes a biochemical analysis of the proteins involved in the formation of pre-replication complex (pre-RC) in Archaea. The yeast two-hybrid system was employed to identify protein-protein interactions within the pre-replication complex of two archaeal species Archaeoglobus fulgidus and Aeropyrum pernix. A library of truncated MCM proteins was used to identify a region that appears to be responsible for hexamerisation of MCM. The pre-RC proteins of archaea belong to the AAA+ family of ATPases. AAA+ proteins function as ATP-driven conformational switches that can mediate the assembly, remodelling, or disassembly of protein complexes. Thus, the assembly of the replication initiation complex may involve an ordered sequence of ATP coupled conformational changes. A variety of biochemical techniques were used to investigate the relationship between ATP hydrolysis, DNA binding and protein interactions within the pre-RC of Archaea.