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

嗜热甲烷嗜热杆菌复制起源及其相关蛋白的表征

• 批准号: 0237483
• 负责人: Zvi Kelman
• 金额: --
• 依托单位: University of Maryland Biotechnology Institute
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0237483&HistoricalAwards=false
• 关键词: Characterization; Methanothermobacter; Thermautotrophicus; Origin; Replication

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.

每个增殖细胞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。该研究项目的长期目标是了解真核生物DNA复制的控制机制，该研究项目将特别关注复制的起源，即染色体上开始复制的特定位置。虽然DNA复制在细菌、病毒和噬菌体中已经有了很好的特征，但真核生物中这个过程的许多方面，特别是在起始阶段，仍然是未知的。复杂的真核细胞周期从起源识别的第一步到细胞分裂的最后一步，包含了大量的调控信号和因子，使得对单个组分和事件的研究变得困难。一种可能有助于规避真核生物复制过程的复杂性的生物是具有真核复制过程的原核生物，即古细菌热自养甲烷菌（Mth）。古细菌的DNA复制似乎更类似于真核生物而不是细菌的过程。然而，古细菌DNA复制装置似乎需要较少的蛋白质和复合物。因此，古细菌可以为理解真核生物复制的酶学提供一个很好的模型系统。本研究将重点探讨Mth中DNA复制起始的机制。野生型和突变型重组蛋白都将用于体外实验，旨在：1)分离和鉴定起源结合蛋白；2)了解起源如何被起始蛋白识别和结合；3)鉴定古细菌起源的功能基序。一个长期的目标是开发一个体外复制系统；从这些生物化学研究中获得的信息将直接适用于这样一个系统。