EXISTENCE OF TORSIONAL TENSION IN EUKARYOTIC DNA IN VIVO

真核和原核染色体中的DNA被组织成 超卷拓扑域。在原核生物中，大约有一半的超级线圈 在DNA中以不受约束的扭转张力来平衡。这样的菌株 已被证明在控制基因表达的机制中起着重要作用。 在真核生物中，大多数超螺旋都受到核小体的限制。 平均而言，DNA螺旋没有受到扭转张力的影响。尽管在那里 没有直接证据表明真核DNA中存在不受限制的超螺旋，一些 目前的基因调控模型表明，扭曲应变的螺旋是 牵涉其中。一个模型表明，真核生物中的扭转张力 染色质可能是由RNA聚合酶在 抄写。扭转张力可以促进呼吸DNA， 形成十字形，形成三链DNA，或形成 左撇子Z-DNA。该提案旨在确定扭矩是否 真核DNA中存在着张力。我们起源的体内试验 无拘无束的扭转张力涉及测量 三甲基补骨脂素与DNA的光结合。这提供了一个平均数 测量不会检测到扭转张力，如果存在于小型 染色体的一部分，如在活跃的基因中或在特定的 DNA中的本地化位置。这项化验将被修改以量化比率。 补骨脂素与选定的DNA限制性片段的交联性 测量选定DNA区域内的扭转应变。我们会 十字形和Z-DNA的合成、表征及应用 活体内自由超螺旋的探头。这涉及到应用 我们开发的基于补骨脂素的分析方法区分了 活细胞中潜在十字形和Z-DNA序列的构象。 这些交替的螺旋结构来自精确的最小(调谐) 允许测量超螺旋水平的超螺旋密度 活体在DNA中的特定位置。将应用交联法 在体内确定SV40微染色体的选定区域或各种 果蝇基因在生活中缠绕着无拘无束的扭转张力 细胞。此外，我们还将插入“扭转调谐探头”序列 进入SV40 DNA的控制区，在可诱导基因的两端 一个稳定的myc表型染色体载体，与果蝇HSP70热相邻 令人震惊的基因询问是否可以检测到自由的DNA超螺旋 构成的或与体内转录有关的。

DNA in both eukaryotic and prokaryotic chromosomes is organized into supercoiled topological domains. In prokarytotes about half the supercoils are equilibrated as unrestrained torsional tension in the DNA. Such strain has been shown to be important in mechanisms controlling gene expression. In eukaryotes, most supercoils are restrained by nucleosomes and,on average, the DNA helix is not wound with torsional tension. Although there is no direct evidence for unrestrained supercoiling in eukaryotic DNA, some current gene regulatory models suggest that a torsionally strained helix is involved. One model suggests that torsional tension in eukaryotic chromatin may be generated by the movement of RNA polymerase during transcription. Torsional tension could promote breathing the DNA, formation of cruciforms, formation of triple stranded DNA, or formation of left-handed Z-DNA. The proposal is designed to determine if torsional tension exists in eukaryotic DNA. Our origin in vivo assay for unrestrained torsional tension involved measurement of rates of trimethylpsoralen photobinding to DNA. This provided an averaging measurement which would not detect torsional tension if present in a small fraction of the chromosome, such as in active genes or at a specific localized site in the DNA. This assay will be modified to quantitate rates of psoralen cross-linking to selected restriction fragments of DNA to measure torsional strain within a selected region of DNA. We will synthesize, characterize, and apply cruciform and Z-DNA "torsionally tuned probes" for unrestrained supercoiling in vivo. This involves application of psoralen-based assays we have developed that distinguish the conformation of potential cruciforms and Z-DNA sequences in living cells. These alternate helical structures from at precise minimum (tuned) superhelical densities allowing measurement of the level of supercoiling in vivo at specific sites in the DNA. The cross-linking assay will be applied in vivo to determine if selected regions of SV40 minichromosomes or various Drosophila genes are wound with unrestrained torsional tension in living cells. In addition we will insert the "torsionally tuned probe" sequences into the control region of SV40 DNA, on either end of an inducible gene in a stable myc epichromosomal vector, and adjacent to Drosophila hsp70 heat shock genes to ask if unrestrained DNA supercoiling can be detected constitutively or in association with transcription in vivo.