TOPOISOMERASE DRUG ACTIONS AT NUCLEAR MATRIX DNA DOMAIN

描述：(申请人摘要)DNA复制的核基质模型 和组织将在拟议的研究中使用，以解决(A) 拓扑异构酶活性药物在初级和高级中的重要作用 染色质结构和(B)细胞对这些药物效应的反应(例如， DNA修复、染色质解体)影响药物作用。中环 有待检验的假设是拓扑异构酶I和II不可逆转地毒害 在复制过程中优先诱导双链DNA断裂损伤DNA 人类CEM细胞的核基质上的叉子。分裂的复制叉子 然后从核基质中分离出来，这会干扰 DNA蛋白激酶(DNA-PK)和复制蛋白A(RPA)系统发出信号 DNA损伤控制和复制分叉修复。ITS的优先切割 不同核基质DNA环区中的c-myc和β-珠蛋白基因将 使用聚合酶链式反应-停止试验(特定目标1)进行定量。这些研究描述了 在具体目标2a中，将通过评估一些 药物引起的神经节细胞脱离的潜在重要生物学后果 C-myc和β-珠蛋白Okazaki片段和复制的DNA环 核矩阵。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 DNA循环。为评价VM-26和喜树碱对卵裂的修复作用， 药物诱导的卵裂在复制和复制过程中的逆转速度 非复制c-myc和β-珠蛋白基因将在CEM和VM-26中被确定 耐药的VM-1细胞。我们还设计了一些额外的实验来量化 药物诱导的RPA32过度磷酸化和Ku70-DNA-PKcs结合的程度 复制和非复制的DNA。这些研究将决定是否 DNA-PK-RPA系统对复制分叉抑制和 双链DNA断裂修复与细胞的逆转程度有关 药物诱导这些细胞系中的DNA裂解。拟议的研究代表了一个 一种新的方法，用于深入了解该病毒的重要生物学效应 拓扑异构酶I和拓扑异构酶II药物及其细胞反应 这些效应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.