Project Summary My lab studies the molecular biology of archaeal microorganisms. Our rationale for doing so lies in the evolutionary relationship between archaea and eukaryotes. Archaeal possess information machineries that are in essence a simplified, ancestral version of the eukaryotic machinery. Viewing these machineries through the prism of archaea provides a unique perspective on the fundamentally conserved molecular mechanisms of genome duplication and expression. We have investigated these processes for many years and have in vitro systems in place for both DNA replication and gene transcription. More recently, we have discovered that archaea of the order Sulfolobales organize their genomes with a compartmentalized architecture – reminiscent of that seen in higher eukaryotes. Our work has revealed a key role for a simple, small SMC-superfamily protein, termed ClsN, in establishing and maintaining this compartmentalized architecture. We are now ideally poised to integrate our chromosome architecture studies with our DNA replication work, to investigate the interplay between genome organization and the dynamic processes of DNA replication and gene expression through combined in vitro, single-molecule and in vivo approaches. With regard to gene expression, we have recently described an unanticipated role for ClsN in effecting an epigenetic state in Sulfolobus. We will dissect the mechanistic basis of this state, determine how applicable it is to other regulatory systems in Sulfolobus and investigate the mechanistic basis of the multi- generational inheritance of ClsN-mediated conformational marks. Taken together, our work will reveal novel insights into the core molecular biology of archaea. Additionally, due to the relationship between archaea and eukaryotes, our work will provide a novel perspective on the evolution and core mechanisms of orthologous processes in eukaryotes.