DNA MISMATCH REPAIR--LESION REPAIR AND STRAND TARGETING

DNA 错配修复——损伤修复和链靶向

• 批准号: 6126997
• 负责人: JAMES T DRUMMOND
• 金额: $ 18.24万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6126997
• 关键词: DNA damage; DNA methylation; DNA repair; HeLa cells; adduct; circular DNA; cis platinum compound; doxorubicin; frameshift mutation; guanine analog; intermolecular interaction; nucleic acid repetitive sequence; plasmids

Our broad, long-term goal is to define the molecular mechanisms by which the human DNA mismatch repair (MR) pathway processes DNA adducts and directs repair to one strand. When certain damaged DNA bases, such as those that result from chemotherapy with cisplatin or alkylating agents, are processed as mismatches, they can induce apoptosis. Clinically significant drug resistance is conferred when MR is silenced. We will define the molecular mechanism by which two of the most effective anticancer drugs, cisplatin and adriamycin, interact with the mismatch repair pathway. In addition, we have developed a new, straightforward method for constructing plasmids that contain a mismatch and a single nick in either DNA strand. This method will be extended to replace mismatches with one of several DNA modifications. Using this approach, we will characterize the mechanism by which covalent DNA damage is recognized and processed by MR. DNA modifications will include cisplatin, transplatin, O6- methylguanine, and 8-oxoguanine. An essential aspect of defining how DNA damage is processed is understanding how strands are distinguished. We will test several possible models by which MR can be targeted to one DNA strand. First, hemimethylation will be tested by constructing mismatch-containing molecules where one strand is selectively methylated at CpG-containing sequences by annealing fully-methylated homoduplex molecules with unmethylated, single-stranded partners. Assays will be performed in vitro, using nuclear extracts from MR-competent HeLa cells. Both covalently closed relaxed and supercoiled molecules will be tested. If hemimethylation is competent to direct MR, we will define the minimal sequence requirements for strand discrimination. We will also ask whether strand gaps or DNA ends can target MR. Free DNA ends, in particular the DNA end in the active site of replicative polymerases, have been proposed to target MR. We will test this model by asking whether free DNA ends, or defined gaps within circular DNAs, are capable of directing MR to the discontinuous strand.

我们广泛的长期目标是确定人类DNA错配修复（MR）途径处理DNA加合物并指导修复一条链的分子机制。 当某些受损的DNA碱基，如顺铂或烷化剂化疗产生的DNA碱基，被处理为错配时，它们可以诱导细胞凋亡。 当MR沉默时，赋予临床显著的耐药性。 我们将定义两种最有效的抗癌药物顺铂和阿霉素与错配修复途径相互作用的分子机制。 此外，我们已经开发了一种新的，简单的方法来构建质粒，包含一个错配和一个单一的切口在任何一个DNA链。 这种方法将被扩展到用几种DNA修饰中的一种来替换错配。使用这种方法，我们将表征的机制，共价DNA损伤的识别和处理MR。DNA修饰将包括顺铂，transplatin，O 6-甲基鸟嘌呤，和8-oxoguanine。 定义DNA损伤如何处理的一个重要方面是了解链是如何区分的。 我们将测试几种可能的模型，通过这些模型，MR可以靶向一条DNA链。 首先，将通过构建含有错配的分子来测试半甲基化，其中通过使完全甲基化的同源双链分子与未甲基化的单链配偶体退火，使一条链在含有CpG的序列处选择性甲基化。 将使用MR感受态HeLa细胞的核提取物在体外进行试验。将测试共价闭合的松弛分子和超螺旋分子。 如果半甲基化能够指导MR，我们将定义链区分的最低序列要求。 我们还将询问链间隙或DNA末端是否可以靶向MR。游离DNA末端，特别是复制型聚合酶活性位点中的DNA末端，已被提议靶向MR。我们将通过询问游离DNA末端或环状DNA内的限定间隙是否能够将MR引导至不连续链来测试该模型。