Repair of Genome Destabilizing DNA Structures

描述（由申请人提供）：已知易位参与许多癌症的病因学，DNA双链断裂（DSB）是许多致癌易位形成的重要第一步。然而，在理解癌基因断裂“热点”处DSB产生的机制方面存在根本性的差距。例如，染色体断裂经常发生在基因组“热点”，并可能导致易位相关疾病。有趣的是，染色体断裂点“热点”通常定位在能够采用替代结构DNA的重复DNA序列附近（即，非B DNA，如H-DNA和Z-DNA），这些结构与癌症病因学有关。申请人的实验室发现，天然存在的Z-DNA（在BCL-2中的断点“热点”处发现）和H-DNA（在c-MYC中的易位断点“热点”附近发现）具有高度致突变性，并且可以在哺乳动物细胞和小鼠中诱导DSB，为进一步测试以下新假设的基本原理提供了基础：1）非B DNA结构被识别为“损伤”并由DNA修复蛋白加工; 2）非B DNA呈现对DNA和RNA聚合酶的阻断，导致DSB;和3）非B DNA诱导的遗传不稳定性在哺乳动物中随着年龄的增长而增加，与易位相关的癌症病因学有关。这项更新申请的长期目标是确定癌症病因学中DNA结构诱导的遗传不稳定性的机制，阐明癌症相关易位的机制，并开发新的方法来减少遗传不稳定性以预防和/或治疗癌症。本申请的直接目标是阐明癌症相关c-MYC和BCL-2基因中染色体断裂的机制，并确定DNA修复、复制和转录在DNA结构诱导的基因组不稳定性中的作用。为了实现我们的目标，我们将使用人类细胞和新型突变报告小鼠来实现以下目标：1）确定DNA修复途径在癌症相关的c-MYC和BCL-2基因中发现的非B DNA的致突变潜力中的作用; 2）阐明人类细胞中非B DNA结构的非复制依赖性和复制依赖性加工机制; 3）评估转录对人类细胞中非B DNA诱导的遗传不稳定性的影响;和4）评估衰老对转基因小鼠组织中人类c-MYC和BCL-2基因的非B DNA形成序列的致突变潜力的影响。这项工作是创新的，因为它将测试新的假设，即在没有DNA损伤的情况下，DNA结构本身被修复机制识别和处理，并且这些结构与癌症病因学有关。拟议研究的预期贡献是阐明癌基因中染色体断裂的机制，以更好地了解癌症病因，这是重要的，因为结果将有助于开发新的策略，以帮助降低癌症发病率。

DESCRIPTION (provided by applicant): Translocations are known to be involved in the etiology of many cancers, and DNA double-strand breaks (DSBs) are an essential first step in the formation of many oncogenic translocations. However, there is a fundamental gap in understanding the mechanisms involved in the generation of DSBs at breakage "hotspots" in oncogenes. For example, chromosomal breakages frequently occur at genomic "hotspots" and can result in translocation-related disease. Interestingly, chromosomal breakpoint "hotspots" are often mapped near repetitive DNA sequences capable of adopting alternatively structured DNA (i.e., non-B DNA, such as H-DNA and Z-DNA), implicating these structures in cancer etiology. The discoveries from the applicant's laboratory that naturally occurring Z-DNA (found at a breakpoint "hotpspot" in BCL-2) and H-DNA (found near a translocation breakpoint "hotspot" in c-MYC) are highly mutagenic and can induce DSBs in mammalian cells and in mice, provide the basis for the rationale going forward to test the novel hypotheses that: 1) non-B DNA structures are recognized as "damage" and are processed by DNA repair proteins; 2) non-B DNA presents a block to DNA and RNA polymerases resulting in DSBs; and 3) non-B DNA-induced genetic instability increases with age in mammals, relevant to translocation-related cancer etiology. The long-term goals of this renewal application are to determine the mechanisms of DNA structure-induced genetic instability in cancer etiology, elucidate the mechanisms involved in cancer-associated translocations, and develop novel approaches to reduce genetic instability to prevent and/or treat cancer. The immediate objectives of this application are to elucidate the mechanisms of chromosomal breakage in the cancer-related c-MYC and BCL-2 genes, and to determine the roles of DNA repair, replication, and transcription in DNA structure-induced genomic instability. To accomplish our goal, we will use human cells and novel mutation-reporter mice in the following aims: 1) determine the roles of DNA repair pathways in the mutagenic potential of non-B DNA found in the cancer- related c-MYC and BCL-2 genes; 2) elucidate the mechanisms of replication-independent and replication- dependent processing of non-B DNA structures in human cells; 3) assess the effects of transcription on non-B DNA-induced genetic instability in human cells; and 4) evaluate the effects of aging on the mutagenic potential of non-B DNA-forming sequences from the human c-MYC and BCL-2 genes in tissues of transgenic mice. The proposed work is innovative because it will test the novel hypothesis that DNA structure, in the absence of DNA damage per se, is recognized and processed by the repair machinery, and that these structures are involved in cancer etiology. The expected contribution of the proposed research is to elucidate the mechanisms of chromosomal breakage in oncogenes to better understand cancer etiology, which is significant because the results will aid in the development of new strategies to help reduce cancer incidence.