ANTICANCER DRUG INDUCED DNA DAMAGE AND REPAIR

这个项目的主要目标是分析 位于小沟或大沟的单部位DNA损伤 的DNA被修复了。为了实现这一目标，我们将构建一个 次要基因内诱导的单位点DNA链间交联 环丙基吡咯吲哚类似物U-77,779(U-77)对DNA的刻槽。这 试剂可诱导跨越两个腺嘌呤N3位的DNA ISC，6 在5‘-TAAAAA-3’或5‘-TAAAAA-3’或5‘- TAAAAAA-3‘序列。DNA ISC位于5‘-GC-3’内的主槽中， 5‘-GNC-3’和5‘-GNNC-3’序列将通过孵育 用两种不同的氮杂二苯二酚定义DNA寡核苷酸 存在两种电子还原酶DT-黄递酶(DTD)。DNA ISC 将通过凝胶电泳法和单点DNA法分离纯化 ISC可以在定义的DNA模板内构建。生物化学 人类肿瘤细胞提取物修复这种损伤的机制将 然后通过三种不同的方式进行评估。1)毁伤能力 与每个单独病变相关联的识别蛋白(DRP)将 要下定决心。然后，这些蛋白质将被分离、纯化和 已确认身份。2)细胞提取液或纯化的酶的能力 切除这类明确的病变将被确定。这将使我们能够 评估碱基切除或核苷酸切除的重要性 这些不同的DNA加合物群。3)细胞提取液的能力 支持对我们定义的受损DNA模板进行修复合成 被评估。后两种DNA修复试验的优点是 可以进行互补性实验。这是一种 修复精通细胞提取液、纯化蛋白或部分纯化 提取物可以补充细胞提取物缺乏的修复活性 DNA修复。这样的实验将对识别和识别 纯化修复DNA ISC所必需的蛋白质 DNA的小沟或大沟。这项工作可能具有重要的意义 在提高我们对以下机制的认识方面的意义 人类肿瘤细胞对DNA反应性抗肿瘤药物具有耐药性。

The major goal of this project is to analyse the mechanisms by which single site DNA lesions located either in the minor groove or major groove of DNA are repaired. In order to achieve this goal we will construct a single site DNA interstrand crosslink (ISC) induced within the minor groove of DNA by the cyclopropylpyroloindole analog U-77,779 (U-77). This agent can induce DNA ISC's which span two adenine N3 positions, 6 nucleotide or 7 nucleotides apart within either a 5'-TAAAAA-3' or 5'- TAAAAAA-3' sequences. DNA ISC located in the major groove within 5'-GC-3', 5'-GNC-3' and 5'-GNNC-3' sequences will be synthesized by incubating a defined DNA oligonucleotide with two distinct aziridinylbenzoquinones in the presence of the two electron reductase DT-diaphorase (DTD). DNA ISC will be isolated and purified by gel electrophoresis and single site DNA ISC can be constructed within a defined DNA template. The biochemical mechanisms by which human tumor cell extracts can repair such lesions will then be assessed in three distinct ways. 1) The ability of damage recognition proteins (DRP's) to associate with each individual lesion will be determined. Such proteins will then be isolated, purified and identified. 2) The ability of either cell extracts or purified enzymes to incise such defined lesions will be determined. This will allows us to assess the importance of either base excision or nucleotide excision to these distinct group of DNA adducts. 3) The ability of cell extracts to support repair synthesis upon our defined damaged DNA templates will also be assessed. The advantages of the latter two DNA repair assays are that complementation experiments can be performed. That is the mixture of a repair proficient cell extract, purified protein or partially purified extract may complement the repair activity of a cell extract deficient in DNA repair. Such experiments will be important for identifying and purifying proteins essential to the repair of DNA ISC located in either the minor or major groove of DNA. This work could have important implication in terms of enhancing our knowledge of the mechanisms by which human tumor cells are resistant to DNA reactive antitumor agents.