Homology-Dependent/Independent Repair of DNA Interstrand

双官能性烷基化剂是许多化疗方案的重要组成部分，可形成多种DNA损伤，包括单加合物、链内和链间DNA交联物(ICL)。链间交链是最有害的损伤之一，使人想起DNA双链断裂，因为它们对DNA复制、重组和基因转录构成了绝对的阻断。虽然修复单加合物和链内交联的机制已经被广泛研究，但由于缺乏病变特异性，对人类链间交联修复的组成和机制知之甚少。 化验。我们已经开发了基于报告重新激活的方法来专门研究哺乳动物细胞中DNA交联链的修复过程。我们的假设是，DNA链间交链的处理可能涉及同源依赖和独立两种机制。我们完成的研究为人类细胞中ICL的去除提供了重组不依赖的机制的证据。在目前的提案中，我们希望实现三个目标。1.确定转录和复制对同源非同源非依赖性ICL修复的影响；2.通过建立基因沉默模型，探讨人类病变旁路聚合酶Rev3在ICL修复中的作用。3.建立ICL同源重组修复的体内模型。这些研究应该会对哺乳动物细胞中DNA链间交联的修复机制产生有益的结果，并使我们能够更好地了解DNA烷基化化疗药物的突变后果。

Bi-functional alkylating agents are important components of many chemotherapeutic regimens that form a variety of DNA damages including mono-adducts, intra- and interstrand DNA cross-links (ICL). Interstrand cross-links are among the most deleterious lesions reminiscent of DNA double strand breaks, because they constitute absolute blockades to DNA replication, recombination, and gene transcription. While mechanisms for repairing mono-adducts and intrastrand cross-links have been studied extensively, little is known about the components and the mechanisms of interstrand cross-linking repair in humans due to the lack of lesion-specific assays. We have developed reporter reactivation-based approaches to examine specifically the repair processes of DNA cross-links in mammalian cells. Our hypothesis is that both homology-dependent and -independent mechanisms may be involved in the processing of DNA interstrand cross-links. Our accomplished studies have provided evidence for a recombination-independent mechanism for ICL removal in human cells. In the present proposal, we will like to accomplish three goals. 1. Determine the effect of transcription and replication on homology-independent ICL repair; 2. Explore the role of human lesion-bypass polymerase Rev3 in ICL repair through generation of gene silencing models. 3. Establish an in vivo model for the homologous recombinational repair of ICLs. These studies should generate informative results on the repair mechanisms of DNA interstrand cross-links in mammalian cells and allows us to better understand the mutagenic consequences of DNA alkylating chemotherapeutic agents.