DNA inverted repeats as an at-risk motif for palindromic gene amplification

描述(申请人提供)：我们研究的目标是确定癌细胞中基因扩增的启动机制。确定启动机制应该提供重要的知识，因为扩增经常驱动肿瘤的进展和治疗抵抗。在基因扩增的早期阶段，一个重要的染色体结构是大染色体区域的反向复制。这表明以下过程(断裂-融合-桥周期，BFB周期)是一种潜在的起始机制；(1)染色体断裂导致复制后断裂末端的融合(姐妹染色单体融合)，从而产生具有两个着丝粒的从头回文(反向复制)染色体；(2)随后的有丝分裂导致两个着丝粒之间的张力(桥)，导致部分复制的染色体末端断裂(断裂)；以及(3)断裂末端进入另一个周期。因此，最初的步骤是一个潜在的限速步骤，因为一旦回文双着丝粒染色体产生，它将不可避免地进入一个糟糕的螺旋(BFB循环)，并导致特定基因组区域的积累(回文基因扩增)。在这个提案中，我们将定义顺式(基因组)和反式(遗传)作用因子，它们在人类肿瘤回文基因扩增的启动中起重要作用。我们之前已经在哺乳动物细胞模型中表明，基因组中预先存在的DNA反向重复序列(DNA-IR)与相邻的DSB一起，极大地促进了回文基因的扩增。因此，基因扩增的第一步是DSB启动的DNA-IR重复之间的非法重组。这导致了我们的DNA水平的模型；DNA-IR中的折叠(链内)退火将产生带有发夹末端的染色体，随后的DNA复制将完成回文复制。基于我们的模型，我们将测试回文基因扩增的潜在决定因素：人类基因组中先前存在的DNA-IR是顺式作用(基因组)决定因素(目标1)，处理发夹末端的基因和诱导DSB的生理条件是重要的反式作用决定因素(目标2)。为了实现我们的目标，我们建立了独特的实验系统，可以克服回文DNA研究中的困难。基因组方法使用了一种新的技术来丰富回文DNA，并确定了用于原发人类肿瘤回文基因扩增的重要DNA-IR。我们的细胞培养系统旨在测量具有不同遗传背景的细胞在DNA-IR上发生DSB启动的非法重组的情况。这些实验应该共同确定特定的DNA-IR作为发展基因扩增的风险基序。考虑到正常人类基因组结构变异的影响，多态DNA-IR可能是一个重要的预测个体对基因扩增易感性的指标。 公共卫生相关性： 项目简介我们研究的主要目标是确定在人类癌症中产生基因放大的机制。基因放大是指在癌细胞中积累额外的基因拷贝，通常会导致细胞异常生长和肿瘤的侵袭性行为。因此，我们对潜在机制的识别将有助于更好地了解癌症是如何发展的，并最终将导致开发早期检测患者基因扩增的干预措施。

DESCRIPTION (provided by applicant): The goal of our research is to define the initiation mechanism of gene amplification in cancer cells. Defining the initiation mechanism should provide important knowledge, as amplification very often drives tumor progression and therapy resistance. An important chromosomal structure at a very early step of gene amplification is an Inverted duplication of large chromosomal regions. This indicates a following process (breakage-fusion-bridge cycle, BFB cycle) as a potential initiation mechanism; (1) a chromosome break leads to the fusion of the broken ends after replication (sister chromatid fusion), resulting in a de novo palindromic (inverted duplication) chromosome with two centromeres; (2) subsequent mitosis causes a tension between the two centromeres (bridge), resulting in a partially duplicated chromosome with a broken end (break); and (3) the broken end enters into another cycle. Therefore, the initial step is a potential rate-limiting step, because, once a palindromic dicentric chromosome is generated, it would inevitably enters into a bad spiral (BFB cycle) and leads to the accumulation of specific genomic regions (palindromic gene amplification). In this proposal, we will define cis- (genomic) and trans- (genetic) acting factors that are important in the initiation of palindromic gene amplification in human tumors. We have previously shown in mammalian cell models that a DNA inverted repeat (DNA-IR) pre-existing in the genome, with an adjacent DSB, greatly promotes palindromic gene amplification. Thus, an initial step of gene amplification is DSB-initiated, illegitimate recombination between the repeats of a DNA-IR. This leads to our DNA-level model; fold-back (Intra-strand) annealing within a DNA-IR would generate a chromosome with a hairpin-capped end, and subsequent DNA replication would complete palindromic duplication. Based on our model, we will test potential determinants for palindromic gene amplification: pre-existing DNA-IRs in the human genome are cis- acting (genomic) determinant (Aim 1), and genes that process a hairpin-capped end and physiological conditions that induce DSBs are important trans-acting determinants (Aim 2). To accomplish our aims, we have established unique experimental systems that can overcome the difficulties in studying palindromic DNA. The genomic approach employs a novel technique for the enrichment of palindromic DNA and identifies important DNA-IRs for palindromic gene amplification in primary human tumors. Our cell culture system is designed to measure the occurrence of DSB-initiated illegitimate recombination at a DNA-IR in cells with a variety of genetic backgrounds. These experiments should collectively identify specific DNA-IRs as an At-Risk Motif for developing gene amplification. Given the impact of structural variations in the normal human genome, polymorphic DNA-IRs could be an important predictor of an individual's susceptibility to gene amplification. PUBLIC HEALTH RELEVANCE: Project Narrative The major goal of our research is to identify the mechanisms generating gene amplification in human cancers. Gene amplification refers to the accumulation of extra copies of genes in cancer cells that often drives abnormal cell growth and aggressive behavior of tumors. Therefore, our identification of underlying mechanisms will provide better understanding of how cancers progress, and eventually will lead to the development of interventions for the early detection of gene amplification in patients.