RADIATION INDUCED CHROMATIN CHANGES IN A DEFINED ORIGIN

描述：（改编自研究者摘要）：细胞具有多个 辐射激发的损伤反应途径。 一 p53介导的通路将细胞阻滞在G1晚期，暂时阻止进入 进入S期或使其转向细胞凋亡。 第二种途径阻止G 细胞进行有丝分裂。 然而，这些细胞周期 检查点可以保护在S期的细胞， 复制受损模板的辐射 私家侦探使用了一个2D 凝胶复制子作图策略显示电离辐射特异性 下调二氢叶酸还原酶（DHFR）的启动 起源于CHO细胞，并证明这种S期损伤敏感（SDS） 途径还抑制人细胞中的中激发rDNA起源的起始， 因此必须在整个S期间起作用。 SDS途径 似乎p53-（可能pRB-）独立。 ori-b和or-g起源于 CHO DHFR结构域已被表征，且每个都含有明显 微球菌核酸酶超敏感位点，可能是信号 来源结合蛋白复合物的存在。 该网站目前在 复制前（G1）状态，但在早期起源激发后消失。 S.有人提出，这种蛋白质/DNA相互作用的修饰是 SDS途径的最终步骤，特异性抑制 对辐射的反应。 这可以通过直接修改 来源结合蛋白（或与之相互作用的蛋白质），或通过全局 对染色质结构的影响，间接影响这种活性， 复杂. 在本申请中，对细胞中的染色质进行了全面的分析， ori-beta和ori-gamma基因座，以及DHFR复制子作为一个整体 本文提出 这些实验旨在区分 局部与全局的可能性。 应特异性来源结合蛋白 修改建议，一个杂交筛选方法，用于确定 将进行编码特定蛋白质的cDNA。 应该更加全球化 修改指出，实验，以确定这些是否 改变通过改变复制子与复制子的结合来实现。 核矩阵描述。 长期目标是了解 在分子水平上的SDS途径，从一个确定的起点开始， 效应器并在通路中反向工作。 由于失去了这条途径， 在共济失调中，毛细血管扩张使细胞对电离 辐射，我们希望开发新的辐射敏化策略， 操纵路径。

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.