DNA MISMATCH REPAIR--LESION REPAIR AND STRAND TARGETING

我们广泛的、长期的目标是确定人类DNA错配修复(MR)途径处理DNA加合物并将修复定向到一条链上的分子机制。当某些受损的DNA碱基，如顺铂或烷化剂化疗导致的DNA碱基被处理为不匹配时，它们可能会诱导细胞凋亡。当MR被沉默时，临床上就会出现显著的耐药性。我们将确定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，我们开发了一种新的、直接的方法来构建在两条DNA链中都包含错配和单个缺口的质粒。这种方法将被扩展，用几种DNA修饰中的一种来取代错配。使用这种方法，我们将描述先生识别和处理共价DNA损伤的机制。DNA修饰将包括顺铂、反铂、O6-甲基鸟嘌呤和8-氧鸟嘌呤。定义DNA损伤是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，通过这些模型可以将MR靶向于一条DNA链。首先，将通过构建包含错配的分子来测试半甲基化，其中一条链在含有CpG的序列上选择性地甲基化，方法是将完全甲基化的同源双链分子与未甲基化的单链伙伴进行退火。检测将在体外进行，使用具有MR能力的HeLa细胞的核提取液。无论是共价封闭的松弛分子还是超螺旋分子都将被测试。如果半甲基化有能力指导MR，我们将定义识别链的最小序列要求。我们还将询问链间隙或DNA末端是否可以靶向MR先生。已提出以复制聚合酶活性部位的DNA末端为靶点的游离DNA末端。我们将通过询问环形DNA中的游离DNA末端或定义的间隙是否能够将MR导向不连续的链来测试这一模型。

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.