DNA Replication in Halobacterium sp. NRC-1

盐杆菌sp. NRC-1是研究古细菌和极端微生物的模式系统。这种古菌的DNA复制特别有趣，因为它可以作为真核生物复制的简单模型，而且对古菌的DNA复制了解相对较少。克隆了模型古细菌盐杆菌NRC-1染色体的复制起点，以及大染色体外复制子pNRC100和pNRC200的共同区域。另外的生物信息学分析已经确定了一组可能参与DNA复制的基因。本项目的目的是进一步研究在盐杆菌NRC-1中DNA复制起始中重要的顺式和反式因子。顺式作用区将通过对先前鉴定的自主复制序列（ARS）元件进行详细的随机和定点诱变来研究，以确定基本区域及其功能。结合保守ARS元件序列的蛋白因子将通过生物化学方法在体内和体外鉴定。该项目的第二个目标是确定哪些基因组区域用于启动体内DNA复制，它们的方向性，以及盐杆菌NRC-1基因组中三个复制子的协调调节机制。为了实现这一目标，将使用DNA微阵列和遗传方法。该项目的第三个目标是利用最近改进的基因敲除系统来解决被预测参与DNA复制起始的基因的本质问题。这个项目将为生命第三分支中一个基本的、研究相对较少的过程提供有价值的见解。对微生物的多样性，特别是对极端微生物的多样性，将有更深入的认识，这可能对生物技术产生影响。这个项目将有助于开发生物研究这一重要领域的人力资源，包括培训研究生和通过外联使其他非科学家参与。最终目标是使对古细菌DNA复制的参与者和过程的理解更接近于对细菌和真核模型系统的理解水平。

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.