Impact of Short Inverted Repeats on Genetic Instability at Mutation Hotspots

描述（由申请人提供）：遗传不稳定性是许多人类疾病的标志，包括癌症。基因组中存在“热点”区域，其特别容易受到DNA双链断裂（DSB）的影响，这可导致不稳定性，例如染色体缺失和易位。然而，这些事件背后的机制并不清楚。因此，本研究的长期目标是通过阐明哺乳动物内源性突变热点的遗传不稳定性机制来填补这一知识空白。有趣的是，具有采用替代DNA结构的能力的序列通常与这些突变“热点”共定位。这些替代的DNA二级结构（即非B DNA）可以在重复序列的片段上形成，具有广泛的生物学功能，并且是许多人类疾病的可能致病因素。例如，十字形DNA结构可以在回文或反向重复（IR）序列处形成，并且通常在人类细胞中的缺失和重排事件的基因组“热点”处发现。为了帮助实现我们的长期目标，近期目标是开发一种新的转基因突变报告小鼠模型，以确定替代DNA结构（与染色体断裂热点共定位）对哺乳动物遗传不稳定性的影响。先前，我们发现来自人c-MYC和BCL-2基因中易位断裂点热点的其他天然存在的非B DNA结构（H-DNA和Z-DNA）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，这暗示这些结构与易位相关的疾病病因学有关。最近，我们确定，短的十字形形成序列（28 bp），这是丰富的人类基因组中，是致突变的，刺激哺乳动物细胞中的DSB。然而，我们还没有测试短十字形形成序列在活生物体中的致突变潜力。因此，在这项研究中，我们将测试的假设，短的DNA十字形是在小鼠的组织特异性的方式致突变。具体而言，我们将：1）开发一种新的十字形DNA突变报告基因转基因小鼠模型; 2）确定十字形DNA在转基因小鼠的各种组织中诱导的突变频率和谱。这些研究的结果将有助于阐明哺乳动物内源性突变热点的遗传不稳定性所涉及的分子机制，并将允许开发新的策略来减少DNA结构诱导的遗传不稳定性，以预防和/或治疗疾病。此外，这项工作可能导致识别用于预防和/或治疗易位相关癌症的新靶点。

DESCRIPTION (provided by applicant): Genetic instability is a hallmark of many human diseases, including cancer. There are "hotspot" regions in the genome that are particularly susceptible to DNA double-strand breaks (DSBs) that can result in instabilities such as chromosomal deletions and translocations. However, the mechanisms underlying these events are not clearly understood. Thus, the long-term objective of this study is to fill this gap in knowledge by elucidating the mechanisms involved in genetic instability at endogenous mutation hotspots in mammals. Interestingly, sequences with the capacity to adopt alternative DNA structures often co-localize with these mutation "hotspots". These alternative DNA secondary structures (i.e. non-B DNA) can form on tracts of repeat sequences, have a wide range of biological functions, and are possible causative factors in a number of human diseases. For example, cruciform DNA structures can form at palindromic or inverted repeat (IR) sequences, and are often found at genomic "hotspots" for deletion and rearrangement events in human cells. To assist in achieving our long-term objective, the immediate goal is to develop a novel transgenic mutation-reporter mouse model to determine the impact of alternative DNA structures (that co-localize with chromosome breakage hotspots) on genetic instability in mammals. Previously, we found that other naturally occurring non- B DNA structures (H-DNA and Z-DNA) from translocation breakpoint hotspots in the human c-MYC and BCL-2 genes, are highly mutagenic and can induce DSBs in mammalian cells and in mice, implicating these structures in translocation-related disease etiology. Recently, we determined that short cruciform-forming sequences (28 bp), which are abundant in the human genome, are mutagenic and stimulate DSBs in mammalian cells. However, we have not yet tested the mutagenic potential of short cruciform-forming sequences in a living organism. Thus, in this study, we will test the hypothesis that short DNA cruciforms are mutagenic in a tissue-specific fashion in mice. Specifically, we will: 1) develop a novel cruciform DNA-mutation reporter transgenic mouse model; and 2) determine the cruciform DNA-induced mutation frequencies and spectra in various tissues from transgenic mice. Results from these studies will assist in elucidating the molecular mechanisms involved in genetic instability at endogenous mutation hotspots in mammals, and will allow the development of novel strategies to reduce DNA structure-induced genetic instability to prevent and/or treat disease. Additionally, this work may result in the identification of novel targets for the prevention and/or treatment of translocation-related cancers.