Halobacterium sp. NRC-1 is a model system for studies of Archaea and extremophiles. DNA replication is of special interest in this Archaeon because it can serve as a simple model for replication in eukaryotes and because it is relatively poorly understood in Archaea. The replication origins from the chromosome of the model archaeaon, Halobacterium NRC-1, and the common region of the large extrachromosomal replicons, pNRC100 and pNRC200 have been cloned. Additional bioinformatic analysis has identified a set of likely genes involved in DNA replication. The goal of this project is to further examine the cis and trans acting factors that are important for DNA replication initiation in Halobacterium NRC-1. The cis acting regions will be studied by detailed random and site-directed mutagenesis of previously identified autonomously replicating sequence (ARS) elements, in order to establish the essential regions and their functions. The protein factors which bind to the conserved ARS element sequences will be identified by biochemical methods, in vivo and in vitro. A second goal of the project is to establish which genomic regions are used to initiate DNA replication in vivo, their directionality, and the mechanism of coordinate regulation of the three replicons in the Halobacterium NRC-1 genome. For this goal, DNA microarrays and genetic methodologies will be used. A third goal of the project is to address the essentially of genes predicted to be involved in the initiation of DNA replication using a recently improved genetic knockout system. This project will provide valuable insights into a fundamental and relatively poorly studied process in the third branch of life. A deeper appreciation of the diversity of microbes, especially extremophiles, will result, with possible implications for biotechnology. This project will contribute to the development of human resources in an important area of biological research, including the training of graduate students and involvement of other non-scientists through outreach. The ultimate goal is to bring the understanding of the players and processes of DNA replication in an archaeon closer to the level of understanding in bacterial and eukaryotic model systems.
