TOPOISOMERASE DRUG ACTIONS AT NUCLEAR MATRIX DNA DOMAIN

描述：（申请人摘要）DNA复制的核基质模型 拟议的研究将利用组织来解决（a） 拓扑异构酶活性药物对初级和高级的重要影响 染色质结构和 (b) 细胞对这些药物作用的反应（例如， DNA 修复、染色质解体）影响药物作用。中央 要检验的假设是拓扑异构酶 I 和 II 不可逆地中毒 通过在复制过程中优先诱导双链 DNA 断裂来损伤 DNA 人类 CEM 细胞核基质上的叉子。分裂的复制叉 然后与核基质分离，这会干扰核基质的能力 DNA 蛋白激酶 (DNA-PK) 和复制蛋白 A (RPA) 系统发出信号 DNA 损伤控制和复制叉修复。其优先裂解 各种核基质 DNA 环域内的 c-myc 和 β-球蛋白基因将 使用 PCR 终止测定（具体目标 1）进行定量。研究描述 在具体目标 2a 中，将通过评估一些内容来扩展这些观察结果 药物引起的分离的潜在重要生物学后果 c-myc 和 beta-球蛋白冈崎片段和复制 DNA 环 核基质。具体目标 2b 和 2c 旨在评估一些 细胞对 VM-26 和喜树碱的反应诱导基质附着损伤 DNA 环。为了评估 VM-26 和喜树碱诱导的裂解的修复， 复制和复制过程中药物诱导的裂解的逆转率 非复制性 c-myc 和 beta-珠蛋白基因将在 CEM 和 VM-26 中测定 耐药VM-1细胞。额外的实验旨在量化 药物诱导的 RPA32 过度磷酸化和 Ku70-DNA-Pkcs 结合的程度 复制和非复制DNA。这些研究将决定是否 DNA-PK-RPA 系统复制叉停滞的激活程度和 双链DNA断裂修复与逆转的程度有关 这些细胞系中药物诱导的 DNA 裂解。拟议的研究代表了 提供洞察重要生物效应的新方法 拓扑异构酶 I 和拓扑异构酶 II 药物以及细胞反应 这些作用可能会影响药物的细胞毒性。

DESCRIPTION: (Applicant's Abstract) The nuclear matrix model of DNA replication and organization will be utilized in the proposed studies to address (a) important effects of topoisomerase-active drugs on primary and higher order chromatin structure and (b) the cellular responses to these drug effects (e.g., DNA repair, chromatin disorganization) that influences drug action. The central hypothesis to be tested is that topoisomerase I and II poisons irreversibly damage DNA by preferentially inducing double-strand DNA breaks in replication forks on the nuclear matrix of human CEM cells. The cleaved replication forks then detach from the nuclear matrix, which interferes with the ability of the DNA protein kinase (DNA-PK) and replication protein A (RPA) systems to signal DNA damage control and replication fork repair. Preferential cleavage of its c-myc and beta-globin genes within various nuclear matrix DNA loop domains will be quantitated using the PCR-stop assay (Specific Aim 1). The studies described in Specific Aim 2a will extend these observations by evaluating some potentially important biological consequences of drug-induced detachment of c-myc and beta-globin Okazaki fragments and replicating DNA loops from the nuclear matrix. Specific Aims 2b and 2c are designed to assess some of the cellular responses to VM-26 and camptothecin induced damage to matrix-attached DNA loops. To evaluate the repair of VM-26 and camptothecin induced cleavage, the rates of reversal of drug-induced cleavage in replicating and nonreplicating c-myc and beta-globin genes will be determined in CEM and VM-26 resistant VM-1 cells. Additional experiments are designed to quantitate the degrees of drug-induced RPA32 hyperphosphorylation and Ku70-DNA-Pkcs binding to replicating and nonreplicating DNA. These studies will determine whether the extent of activation of the DNA-PK-RPA system for replication fork arrest and double strand DNA break repair is related to the degree of reversal of drug-induced DNA cleavage in these cell lines. The proposed studies represent a novel approach for providing insights into important biological effects of the topoisomerase I and topoisomerase II drugs as well as the cellular responses to these effects that may influence drug cytotoxicity.