CAREER: Methanosarcina Acetivorans as a Model to Elucidate the Mechanism of DNA Replication in the Archaea

University Of Illinois At Urbana-Champaign, Urbana IL

Investigators

Abstract

Life on the planet Earth is divided into three domains: Archaea, Bacteria, and Eukarya. Each of the three lineages is propagated through transfer of genetic material by DNA replication, a complex interplay of many proteins acting in concert to duplicate the genome. While significant progress has been made in the elucidation of the mechanisms involved in the replication of bacterial and eukaryotic genomes, the current understanding of genome replication in the archaea is rudimentary. Several genes encoding eukaryotic-like proteins known to be essential at the replication fork have been identified in all archaeal genomes sequenced to date. A few of the proteins have been shown to possess predicted biochemical activities in vitro. However, the direct evidence for the role of these proteins in archaeal cells is missing due to lack of reliable genetic manipulation systems for the hyperthermophiles currently used as models to study archaeal DNA replication. This career project will use Methanosarcina acetivorans, an archeaon with a proven genetic manipulation system and a completely sequenced genome, as a model to decipher the archaeal DNA replication mechanism. Genetic methods will be used to identify genomic DNA fragments capable of autonomous replication in M. acetivorans to determine the archaeal origin/s of replication. The genes coding for the predicted proteins involved in (a) origin recognition, (b) loading of the replicative DNA helicase (c) the switch from origin recognition to initiation of DNA synthesis, (d) elongation and (e) processing of Okazaki fragments will be disrupted in the genome to determine whether they are essential for cell viability. These experiments, together with in vitro biochemical characterization of each protein under study, aside from enhancing the understanding of genome replication in the third major domain of life, will provide a simpler model in the quest to completely unravel the complex eukaryotic DNA replication machinery. The project will provide training for a diversity of graduate and undergraduate students, and minority high school students during summer, in (a) the use of computers to predict protein function, (b) gene cloning and manipulation, (c) protein purification and (d) biochemical characterization all aimed at assigning function to genes. The project thus aims at preparing students to tackle one of the inevitable challenges of the post-genomics era: assignment of function to the ubiquitous hypothetical and conserved proteins in known genome sequences. In addition, the content of the research serves as the teaching material for the microbial functional genomics component of ANSCI 199 (Discovering the Genome) taught to undergraduate students every spring semester.