STABLE AND DYNAMIC DNA SUPERCOILING IN VIVO

这里描述的研究将调查DNA的性质 通过探索两个平行模型在活细胞中进行超螺旋：(A) 超线圈作为一种动态特性，可因DNA的不同而局部变化 跟踪过程，如转录；和(B)作为一种 有助于结构的染色体的稳定性质 基因组的组织和紧凑。目前，量化的 动态超卷与体内稳定超卷的关系 是一个悬而未决的问题。转录可以引起对 染色体的整体超螺旋密度，但效率与 目前还不清楚是哪种RNA聚合酶引入了超级转角。这个 转录改变超螺旋张力的能力取决于许多因素 机制因素，如RNA聚合酶的锚定程度， 超螺旋沿DNA扩散的速度，动力学 拓扑异构酶的周转，以及可能存在的拓扑 染色体上的障碍。这里提出的研究的一部分是 基于其中转录可以展开模板的模型系统 负超螺旋密度大于或等于0.35 几秒钟。这一速度接近伸长的最大效率 RNA聚合酶，其中转录十个碱基对引入一个碱基对 前面是正超级转弯，后面是负超级转弯 转录复合体。在这个模型系统中，一个标记基因的表达 强烈地受到另一个相邻的方向的影响， 强表达基因。在实验上，该系统提供了一种 体内拓扑异构酶的动力学估计。进一步 研究将阐明一些基本的相互作用 细胞中的RNA聚合酶。这项提议的另一个重要因素是 了解真核生物中稳定的DNA超螺旋的本质 直接探针法检测猪染色体上的扭转应变DNA 酵母。提案的这一部分应该能让您深入了解 真核细胞染色体中拓扑域的存在 对DNA超螺旋的维护有重要意义 以及高等生物体中的基因表达。最后，稳定的拓扑图 古细菌染色体的结构和DNA是否 在体内的超高温成员中呈正超螺旋 这一组是这项研究的第三个主要方面。

The research described here will investigate the nature of DNA supercoiling in living cells by exploring two parallel models: (A) Supercoiling as a dynamic property which can vary locally due to DNA tracking processes such as transcription; and (B) Supercoiling as a stable property of chromosomes which contributes to structural organization and compaction of the genome. Currently, the quantitative relationship between dynamic supercoiling and stable supercoiling in vivo is an unresolved question. Transcription can give rise to changes in the overall superhelix density of the chromosome, but the efficiency with which RNA polymerases introduce superturns is not yet understood. The ability of transcription to alter superhelical tension depends on many mechanistic factors such as the degree of anchoring of RNA polymerase, the rate at which supercoils diffuse along DNA, the kinetics of topoisomerase turnover, and the possible existence of topological barriers along the chromosome. Part of the research proposed here is based on a model system in which transcription can unwind the template to a negative superhelix density greater than or equal to 0.35 in tens of seconds. This rate approaches the maximal efficiency for an elongating RNA polymerase wherein transcription of ten basepairs introduces one positive superturn in front of and one negative superturn behind the transcription complex. In this model system, expression of a marker gene is strongly affected by the orientation of another adjacent, strongly-expressed gene. Experimentally, this system provides an estimate of the kinetics of topoisomerase enzymes in vivo. Further studies will elucidate some of the fundamental interactions which anchor RNA polymerase in cells. Another important element of this proposal is to understand the nature of stable DNA supercoiling in eukaryotes by directly probing for torsionally strained DNA on the chromosomes of yeast. This part of the proposal should provide insight into the existence of topological domains in eukaryotic chromosomes which will have important implications for the maintainenance of DNA supercoiling and gene expression in higher organisms. Finally, the stable topological structure of archaebacterial chromosomes and the question of whether DNA is positively supercoiled in vivo in the hyperthermophilic members of this group is a third major aspect of this research.