The Characterization of the Methanothermobacter Thermautotrophicus Origin of Replication and its Associated Proteins

每个增殖细胞都必须准确地复制其遗传信息，并且每个细胞周期只能复制一次。任何低于或超过这些限制的过程都会导致细胞死亡或异常增殖。这项研究计划的长期目标是了解真核生物DNA复制的控制机制，这项研究项目将专门关注复制的起源，即染色体上复制开始的特定位置。虽然DNA复制已经在细菌、病毒和噬菌体中得到了很好的表征，但真核生物中这一过程的许多方面，特别是在起始阶段，仍然未知。从起源识别的第一步到细胞分裂的最后一步，复杂的真核细胞周期涉及众多的调控信号和因素，这使得对单个成分和事件的研究变得困难。一种可能有助于绕过真核生物复制的一些复杂性的生物是具有真核生物样复制过程的原核生物，即古甲烷热营养杆菌(MTH)。古生菌的DNA复制似乎更类似于真核生物，而不是细菌的过程。然而，古生菌DNA复制设备似乎需要较少的蛋白质和复合体。因此，古生菌可能为理解真核生物复制的酶学提供了一个很好的模式系统。这项研究将集中在线粒体DNA复制的启动机制上。野生型和突变型重组蛋白都将用于体外实验，目的是：1)分离和鉴定起始物结合蛋白，2)了解起始物是如何识别和结合起始物的，以及3)确定古生菌起源中的功能基序。一个长期目标是开发一种针对转基因生物的体外复制系统；从这些生化研究中获得的信息将直接适用于这样的系统。

Every proliferating cell must duplicate its genetic information accurately and only once per cell cycle. Any process that falls short of or exceeds these limits results in cell death or abnormal proliferation. The long-term goal of this research program is to understand the mechanisms governing DNA replication in eukarya, and this research project will focus specifically on the origin of replication, a specific location on the chromosome where replication initiates. Whereas DNA replication has been well characterized in bacteria, viruses and bacteriophages, many aspects of the process in eukarya, particularly in the initiation phase, are still unknown. The complex eukaryotic cell cycle entails numerous regulatory signals and factors from the first step of origin recognition to the final step of cell division, making it difficult to study individual components and events. An organism that may help to circumvent some of the complexity of eukaryotic replication is a prokaryote with eukaryotic-like replication processes,the archaeon Methanothermobacter thermautotrophicus (Mth). Archaeal DNA replication appears to be more similar to the eukaryal rather than the bacterial process. However, the archaeal DNA replication apparatus appears to require fewer proteins and complexes. Thus, archaea may provide a good model system for understanding the enzymology of eukaryotic replication. The research will focus on the mechanisms of the initiation of DNA replication in Mth.Both wild-type and mutant recombinant proteins will be used in in vitro experiments designed to:1) isolate and identify the origin binding proteins,2)understand how the origin is recognized and bound by the initiation proteins; and 3) identify the functional motifs in the archaeal origin. A long-term goal is to develop an in vitro replication system for Mth; the information gained from these biochemical studies will be directly applicable to such a system.