DNA MISMATCH REPAIR--LESION REPAIR AND STRAND TARGETING

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加合物并将修复引导到一条链的分子机制。当某些受损的DNA碱基，如顺铂或烷基化剂化疗导致的DNA碱基，被处理为不匹配时，它们可以诱导细胞凋亡。当MR沉默时，具有临床意义的耐药性被授予。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，直接的方法来构建质粒，在DNA链中包含一个不匹配和单个缺口。该方法将扩展到用几种DNA修饰中的一种来替换不匹配。使用这种方法，我们将描述mr识别和处理共价DNA损伤的机制，DNA修饰将包括顺铂、移植、O6-甲基鸟嘌呤和8-氧鸟嘌呤。定义DNA损伤如何处理的一个重要方面是了解如何区分链。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，通过构建含有错配的分子来测试半甲基化，其中一条链在含有cpg的序列上选择性甲基化，通过退火完全甲基化的同双工分子与未甲基化的单链伙伴。实验将在体外进行，使用来自MR-competent HeLa细胞的核提取物。共价闭合松弛分子和超卷曲分子都将被测试。如果半甲基化能够指导MR，我们将定义链识别的最小序列要求。我们还将询问链间隙或DNA末端是否可以靶向MR。自由DNA末端，特别是复制聚合酶活性位点的DNA末端，已被提出用于靶向MR。我们将通过询问自由DNA末端或圆形DNA内定义的间隙是否能够将MR定向到不连续链来测试该模型。

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.