ENZYMATIC PROBES FOR OPEN REGIONS IN SUPERCOILED DNA

超螺旋 DNA 开放区域的酶探针

• 批准号: 2175853
• 负责人: DAVID KOWALSKI
• 金额: $ 6.5万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175853
• 关键词: DNA gyrase; DNA replication; Escherichia coli; Leguminoseae; Z DNA; bacterial DNA; bioenergetics; chemical cleavage; chemical structure function; chromatin; circular DNA; conformation; endonuclease; enzyme mechanism; gel electrophoresis; gene expression; genetic manipulation; genetic transcription; molecular cloning; nucleic acid probes; nucleic acid sequence; nucleic acid structure; plasmids; point mutation; simian virus 40; tissue /cell culture; virus DNA

The long term goal of this proposal is to understand how the structure and energetics of the DNA template contribute to regulatory mechanisms for the initiation of DNA replication and transcription in living cells. Supercoiling of the DNA template is energetically unfavorable and can result in localized opening or unwinding of the helix in vitro. This proposal is a continuation of our characterization of the single-strand- specific endonucleases, mung bean nuclease and P1 nuclease, as probes for unwinding in naturally-occurring DNA sequences present in supercoiled plasmids and viral genomes. The enzymes recognize Z-DNA, cruciforms and a novel, stably-unwound DNA conformation call the AT-rich structure. The AT- rich structure occurs in several prokaryotic and eukaryotic replication origins. Extensive mutational analysis of a yeast replication origin revealed that detection of the AT-rich structure in vitro correlates with the ability to initiate DNA replication in vivo. The specific aims of this proposal are to 1) examine the architecture, DNA sequence selectivity and energetics of the AT-rich structure, 2) investigate easily-unwound DNA sequences as determinants for initiation of replication in vivo, and 3) probe for localized DNA unwinding in chromatin. The hypothesis that the AT-rich structure is not melted but rather a lower energy DNA conformation which is partially untwisted and base paired, will be tested by 1) two dimensional gel electrophoresis of topoisomers to assess the energy and extent of DNA unwinding, and 2) analysis of DNA sequences which react with the enzymes as well as chemical probes for paired or unpaired bases. They hypothesis that the low energy of DNA unwinding is a determinant of replication initiation in vivo will be tested by comparing the effects of mutations on formation of an AT-rich structure in the chromosomal origin (oriC) of E. coil and the simian virus 40 (SV40) origin in vitro with the ability of the mutant derivatives to initiate replication in vivo. Finally, we will establish the use of mung bean nuclease and P1 nuclease as chromatin probes for unpaired and non-Watson-Crick paired bases, in SV40 chromatin at nucleotide level resolution.

该提议的长期目标是了解结构和 DNA模板的能量学有助于调节机制 活细胞中DNA复制和转录的启动。 DNA模板的超涂层在能量上是不利的，可以 导致体外局部开放或放松螺旋。 这 提案是我们对单链的表征的延续 特定的核酸内切酶，绿豆核酸酶和P1核酸酶作为探针 在超螺旋中放松自然出现的DNA序列 质粒和病毒基因组。 酶识别Z-DNA，十字形和A 新颖的，稳定的无链DNA构象称为富含的结构。 AT- 丰富的结构发生在几种原核生物和真核复制中 起源。 酵母复制起源的广泛突变分析 揭示了在体外富含水平结构的检测与 在体内启动DNA复制的能力。 这个特定的目的 建议是1）检查体系结构，DNA序列选择性和 富含富集结构的能量，2）研究易于解开的DNA 序列是在体内启动复制的决定因素，3） 探测染色质中局部DNA的局部DNA。 假设 富裕的结构不是融化的，而是较低的能量DNA构象 部分不介于twist和基础配对，将通过1）测试。 拓扑异构体的尺寸凝胶电泳，以评估能量和 DNA解开的程度，以及2）与DNA序列的分析，这些序列与 酶以及配对或未配对碱基的化学探针。 他们 假设DNA的低能量放松是决定因素 将通过比较 染色体起源中富集结构形成的突变 E. coil的（Oric）和邻苯二甲酸病毒40（SV40）起源于体外 突变衍生物在体内启动复制的能力。 最后，我们将建立绿豆核酸酶和P1核酸酶的使用 在SV40中 核苷酸水平分辨率的染色质。