Analysis of a Stress-Inducible DNA-Binding Protein in a Cyanobacterium

蓝藻中应激诱导 DNA 结合蛋白的分析

• 批准号: 9634049
• 负责人: George Bullerjahn
• 金额: $ 18.64万
• 依托单位: Bowling Green State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9634049&HistoricalAwards=false
• 关键词: Analysis; Stress; Inducible; DNA; Binding

9634049 Bullerjahn This project is an investigation of a mechanism by which the cyanobacterium Synechococcus sp. strain PCC 7942 can adapt to oxidative stress. The gene dpsA, encoding a stress-induced DNA-binding protein has been cloned and sequenced. Previous work has shown that the DpsA protein accumulates under conditions of nutrient stress as well during the stationary phase of the growth curve. The protein forms extremely stable hexameric complexes that bind DNA; studies of the evolutionarily divergent Escherichia coli gene and its product indicate the DpsA protein serves to protect the chromosome from oxidative stress during long-term nutrient depletion and exposure to peroxide. However, a major difference between the E. coli and Synechococcus polypeptides is the presence of a heme-binding domain in the C-terminal half of the Synechococcus DpsA protein. It is suggested that the function of heme is to consume excess peroxide by a catalase mechanism. There is potentially a higher demand for detoxifying reactive species of oxygen in oxygen-producing phototrophs such as cyanobacteria. Mutants defective in the production of DpsA will be examined with respect to their ability to survive conditions of photooxidative damage and peroxide treatment. Truncated and chimeric forms of dpsA will also be introduced into dpsA null mutants of Synechococcus in order to test whether the presence of the heme-binding domain plays an important role in survival. RNA blots and promoter fusions will be used to determine the environmental conditions contributing to dpsA transcription. Immunocytochemical methods will be used to attempt to determine the location of the DpsA/DNA complex in the cells during adaptation to stationary phase. This study may help determine whether there is a distinct structural region of the nucleoid that is preferentially protected by the DpsA protein complex. Lastly, the yeast two-hybrid system will be employed to screen for possible protein-protein interactions that might be involved in regulating assembly and turnover of the stable DpsA complex. Overall, this work will help reveal mechanisms involved in global stress responses in cyanobacteria, and other phototrophs. %%% This study is concerned with how the cyanobacterium Synechococcus sp. strain PCC 7942 can adapt to conditions which cause oxidative damage to cell components. The gene dpsA and the protein it encodes DpsA will be studied. DpsA helps protect the cells against oxidative damage by an unusual mechanism. This proposal will focus on how light and other environmental conditions control production of DpsA. These studies will provide new information about how photosynthetic organisms respond to oxidative stress and change. ***

9634049 Bullerjahn该项目是对蓝细菌聚球藻属菌株PCC 7942能够适应氧化应激的机制的研究。 已克隆并测序了编码胁迫诱导的DNA结合蛋白的基因dpsA。 以前的工作表明，DpsA蛋白积累的营养胁迫条件下，以及在生长曲线的稳定期。 该蛋白质形成非常稳定的六聚体复合物，结合DNA;对进化上不同的大肠杆菌基因及其产物的研究表明，DpsA蛋白在长期营养耗尽和暴露于过氧化物期间保护染色体免受氧化应激。 然而，E.与大肠杆菌和聚球藻多肽的差异在于在聚球藻DpsA蛋白的C-末端一半中存在血红素结合结构域。 有人建议，血红素的功能是通过过氧化氢酶机制消耗过量的过氧化物。 在产氧光养生物如蓝细菌中，对解毒活性氧的需求可能更高。 将检查DpsA产生缺陷的突变体在光氧化损伤和过氧化物处理条件下存活的能力。 截短和嵌合形式的dpsA也将被引入到聚球藻的dpsA无效突变体中，以测试血红素结合结构域的存在是否在存活中起重要作用。 RNA印迹和启动子融合将用于确定有助于dpsA转录的环境条件。 将使用免疫细胞化学方法来尝试确定DpsA/DNA复合物在适应稳定期期间在细胞中的位置。 这项研究可能有助于确定是否有一个独特的结构区域的类核，优先保护的DpsA蛋白复合物。 最后，酵母双杂交系统将用于筛选可能参与调节稳定DpsA复合物的组装和周转的蛋白质-蛋白质相互作用。 总的来说，这项工作将有助于揭示蓝藻和其他光养生物的全球应激反应机制。 本研究关注的是蓝细菌聚球藻属菌株PCC 7942如何能够适应对细胞组分造成氧化损伤的条件。 本研究对dpsA基因及其编码蛋白进行了研究。 DpsA通过一种不寻常的机制帮助保护细胞免受氧化损伤。 该提案将重点关注光线和其他环境条件如何控制DpsA的生产。 这些研究将为光合生物如何应对氧化应激和变化提供新的信息。 ***