TOPOISOMERASE DRUG ACTIONS AT NUCLEAR MATRIX DNA DOMAIN

描述：（申请人的摘要）DNA复制的核基质模型 及组织架构，以处理以下问题： 拓扑异构酶活性药物对初级和高级的重要作用 染色质结构和（B）对这些药物作用的细胞应答（例如， DNA修复，染色质解体），影响药物作用。中央 待检验假设是拓扑异构酶I和II不可逆地 通过优先诱导复制中的双链DNA断裂来损伤DNA 人类CEM细胞核基质上的分叉。切割的复制叉 然后从核基质中分离出来，这会干扰细胞的能力。 DNA蛋白激酶（DNA-PK）和复制蛋白A（RPA）系统 DNA损伤控制和复制叉修复。其优先裂解 不同核基质DNA环结构域中的c-myc和β-珠蛋白基因将 使用PCR终止试验（特异性目的1）进行定量。描述的研究 在具体目标2a中，将通过评估一些 药物诱导的脱离的潜在重要生物学后果 c-myc和β-珠蛋白冈崎片段和复制DNA环从 核基质具体目标2b和2c旨在评估一些 细胞对VM-26和喜树碱诱导的基质附着损伤的反应 DNA循环为了评价VM-26和喜树碱诱导的切割的修复， 逆转药物诱导的切割在复制和 将在CEM和VM-26中测定非复制型c-myc和β-珠蛋白基因 抗VM-1细胞。设计另外的实验以定量 药物诱导的RPA 32过度磷酸化和Ku 70-DNA-PKcs结合的程度 复制和非复制DNA这些研究将确定 用于复制叉停滞的DNA-PK-RPA系统的激活程度， 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 一种新的方法，可以深入了解 拓扑异构酶I和拓扑异构酶II药物以及细胞对 这些效应可能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.