ENZYMATIC PROBES FOR OPEN REGIONS IN SUPERCOILED DNA

超螺旋 DNA 开放区域的酶探针

• 批准号: 3278412
• 负责人: DAVID KOWALSKI
• 金额: $ 11.95万
• 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 1986-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278412
• 关键词: DNA gyrase; Escherichia coli; Leguminoseae; chemical cleavage; chemical structure function; circular DNA; endonuclease; gel electrophoresis; genetic manipulation; molecular cloning; mutant; nuclease; nucleic acid sequence; phosphodiesterases; radiotracer; simian virus 40; virus DNA; virus replication; yeasts

Higher order coiling or supercoiling exists in the double-stranded DNA of all organisms as well as some viruses. Negative supercoiling imparts a torsional strain which can produce open or single-stranded regions at specific sites in DNA. These regions may serve as recognition sites for regulatory proteins and enzymes involved in DNA replication, transcription and recombination and may have other functions. The long-term goal of this proposal is to examine the presence, nature and biological significance of single-stranded regions in supercoiled DNA. To achieve this goal, we will (1) evaluate three different single-strand specific endonucleases as probes for open regions in pBR322 DNA by determining the nucleotide sequences cleaved and the effects of environmental conditions and superhelical density on the site specificity, (2) probe the structure of other supercoiled DNAs to test the generality of our findings, and (3) enzymatically delete single-stranded regions to directly test their biological importance in vivo. Knowledge of the relationships between DNA supercoiling, DNA structure and biological function is essential for understanding the regulation and execution of DNA functions in normal and cancer cells.

高阶盘绕或超螺旋存在于双链的DNA中 所有生物以及一些病毒。 负超螺旋授予 扭转应变，可以在 DNA中的特定位点。 这些地区可能是公认网站 参与DNA复制，转录的调节蛋白和酶 和重组，可能具有其他功能。 这个长期目标 提议是检查的存在，性质和生物学意义 超螺旋DNA中的单链区域。 为了实现这一目标，我们将 （1）作为探针评估三个不同的单链特异性内切酶 通过确定核苷酸序列，用于PBR322 DNA中的开放区域 裂解以及环境条件和超螺旋的影响 位点特异性上的密度，（2）探测其他的结构 超螺旋DNA测试我们发现的一般性，（3） 酶上删除单链区域以直接测试其 体内生物学重要性。 了解DNA之间的关系 超螺旋，DNA结构和生物功能对于 了解正常的DNA功能的调节和执行 癌细胞。