RADIATION INDUCED CHROMATIN CHANGES IN A DEFINED ORIGIN

描述：(改编自调查人员摘要)：细胞有多个 被辐射挑战激活的损伤反应通路。一个 P53介导的通路在G1晚期阻止细胞进入，以暂时阻止进入 进入S期或将其分流至细胞凋亡。第二条途径是预防G 阻止细胞进行有丝分裂。然而，这两种细胞周期 检查站可以保护当时处于S阶段的细胞 复制损坏的模板所产生的辐射。私家侦探用了一个2-D 凝胶复制子图谱策略以显示特定的电离辐射 下调二氢叶酸还原酶(DHFR)早期启动 起源于CHO细胞，并证明了这种S时相损伤传感(SDS) 途径也抑制了人类细胞中rDNA起源的启动， 因此必须在整个S时期发挥作用。十二烷基硫酸钠途径 P53-(可能是pRb-)不依赖于细胞。ORI-B和OR-G起源于 已经表征了CHO DHFR结构域，并且每个结构域都包含一个发音的 微球菌核酸酶超敏部位，可能发出信号 存在与起源结合的蛋白质复合体。该站点位于 复制前(G1)状态，但在早期触发原点后消失 美国提出，这种蛋白质/DNA相互作用的修饰是 在SDS途径中的最后一步，它特异性地抑制 对辐射的反应。这可以通过直接修改 原产地结合蛋白(或与之相互作用的蛋白)，或由全球 对染色质结构的影响，间接影响该酶的活性 很复杂。在这一应用中，对染色质的全面分析 Ori-beta和Ori-Gamma基因座，以及DHFR复制子的整体 是提议的。这些实验是为了区分 本地的可能性与全球的可能性。是否应该有特定的原产地结合蛋白 提出了一种单杂交筛选方法，用于鉴定沙门氏菌 将进行编码特定蛋白的cDNA.应该更加全球化 指示修改，实验以确定这些 更改通过更改复制子与 描述了核基质。长期目标是理解 分子水平上的十二烷基硫酸钠途径，从一个确定的起源开始及其 效应器，并在路径中向后工作。因为失去了这条通路 在共济失调中，毛细血管扩张使细胞对电离极其敏感 辐射，我们希望通过以下方式开发新的辐射敏化策略 对通路的操纵。

DESCRIPTION: (Adapted from investigator's abstract): Cells have multiple damage-response pathways that are activated by a radiation challenge. A p53-mediated pathway arrests cells in late G1, to temporarily prevent entry into S phase or to shunt them toward apoptosis. A second pathway prevents G cells from undergoing mitosis. However, neither of these cell cycle checkpoints can protect cells that are in the S phase at the time of radiation from replicating damaged templates. The P.I. has utilized a 2-D gel replicon mapping strategy to show that ionizing radiation specifically down-regulates initiation in the early firing dihydrofolate reductase (DHFR) origin in CHO cells and demonstrated that this S-phase damage-sensing (SDS) pathway also inhibits initiation in mid-firing rDNA origins in human cells, and therefore must function throughout the S period. The SDS pathway appears p53- (and probably pRB-) independent. The ori-b and or-g origins in the CHO DHFR domain have been characterized, and each contains a pronounced micrococcal nuclease hypersensitive site that presumably signals the presence of an origin-binding protein complex. This site is present in the pre-replicative (G1) state, but disappears after the origin fires in early S. It is proposed that modification of this protein/DNA interaction is the ultimate step in the SDS pathway that specifically inhibits initiation in response to radiation. This could occur by direct modification of an origin-binding protein (or a protein that interacts with it), or by a global effect on chromatin structure that indirectly affects the activity of this complex. In this application, a comprehensive analysis of the chromatin in the ori-beta and ori-gamma loci, as well as in the DHFR replicon as a whole is proposed. These experiments are proposed to distinguish between the local versus global possibilities. Should specific origin-binding protein modification be suggested, a one hybrid screening method for identifying the cDNA encoding the specific protein will be performed. Should more global modifications be indicated, experiments to determine whether these alterations are effected by altering association of the replicon with the nuclear matrix are described. The long-range goals are to understand the SDS pathway at the molecular level, starting at a defined origin and its effectors and working backward in the pathway. Since loss of this pathway in Ataxia Telangiectasia renders cells exquisitely sensitive to ionizing radiation, we hope to develop novel radiation sensitization strategies by manipulation of the pathway.