ENZYMATIC PROBES FOR OPEN REGIONS IN SUPERCOILED DNA

超螺旋 DNA 开放区域的酶探针

• 批准号: 3278414
• 负责人: DAVID KOWALSKI
• 金额: $ 15.09万
• 依托单位: ROSWELL PARK CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 1989-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278414
• 关键词: DNA; Escherichia coli; Leguminoseae; chemical cleavage; chemical structure function; chromosome deletion; circular DNA; endonuclease; enzyme mechanism; gel electrophoresis; gene deletion mutation; gene expression; genetic manipulation; genetic transcription; molecular cloning; nuclease; nucleic acid sequence; nucleic acid structure; plasmids; point mutation; radiotracer; simian virus 40; virus DNA; virus replication; yeasts

Higher order coiling or supercoiling exists in the DNA of all organisms as well as some viruses. The long term goal of this proposal is to understand the structure of supercoiled DNA and the molecular mechanisms by which supercoiling affects biological function. Natural supercoiling can lead to formation of open (unwound or unrepaired) regions in DNA. Such regions are frequently associated with altered DNA secondary structures, eg. cruciforms and Z-DNA. We will continue to use mung bean nuclease, a single-strand-specific endonuclease, to probe for altered secondary structures under solution conditions where DNA can function in vitro. In prokaryotic and eukaryotic DNAs, the enzyme often cleaves DNA sequences involved in regulation of transcription and DNA replication. Potential cruciforms and Z-DNA structures are recognized as is a dA+dT-rich sequence with a novel conformation. The goal of the proposal is to examine the nature and biological significance of altered secondary structures in supercoiled DNA. To achieve this goal we will continue to study plasmid pBR322 DNA and simian virus 40 DNA, and we will (1) examine the effects of point mutations on detection of altered secondary structure, (2) examine the effects of point mutations on gene expression in vivo and transcription in vivo and in vitro, (3) determine the requirements for recognition of a new DNA conformation detected in certain dA-dT-rich sequences. Point mutations will be introduced by either oligonucleotide directed mutagenesis or by gap repair mutagenesis at mung bean nuclease nicks. Recombinant DNA technology and prokaryotic and eukaryotic expression vectors will be utilized to assess gene expression in vivo. The level and accurate initiation of RNA transcripts will be determined by annealing mRNA to labeled complementary DNA and analyzing the DNA protected from single-strand-specific nuclease digestion on DNA sequencing gels.

所有生物的DNA中都存在高阶盘绕或超串联 以及一些病毒。 该提议的长期目标是了解 超螺旋DNA的结构和分子机制 超螺旋影响生物学功能。 天然超螺旋会导致 DNA中开放（解开或未修复的）区域的形成。 这样的地区是 通常与DNA二级结构改变有关，例如十字形 和Z-DNA。 我们将继续使用绿豆核酸酶 单链特异性核酸内切酶，以探测改变的次要 在溶液条件下DNA可以在体外起作用的结构。 在 原核和真核DNA，酶经常切割DNA序列 参与转录和DNA复制的调节。 潜在的 十字形和Z-DNA结构被识别为富含DA+DT的序列 具有新颖的构象。 该提议的目的是检查 二级结构改变的性质和生物学意义 超螺旋DNA。 为了实现这一目标，我们将继续研究质粒 PBR322 DNA和替代病毒40 DNA，我们将（1）检查 检测二次结构改变的点突变，（2）检查 点突变对体内基因表达和转录的影响 体内和体外，（3）确定识别a的要求 在某些DA-DT的序列中检测到的新DNA构象。 观点 任何一个寡核苷酸定向诱变将引入突变 或通过绿豆核酸酶划痕的间隙修复诱变。 重组DNA 技术，原核生物和真核表达向量将是 用于评估体内基因表达。 水平和准确 RNA转录本的启动将通过将mRNA退火确定 标记为互补的DNA并分析受保护的DNA DNA测序凝胶上的单链特异性核酸酶消化。