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

描述（由申请人提供）：我们研究的目的是确定癌细胞中基因扩增的起始机制。定义启动机制应该提供重要的知识，因为扩增通常会导致肿瘤进展和治疗耐药性。在基因扩增的非常早期阶段，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-桥循环，BFB循环）作为一种潜在的启动机制;（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），导致从头回文（反向重复）有两个着丝粒的染色体;（2）随后的有丝分裂引起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（break）;（3）断头进入下一个循环。因此，初始步骤是潜在的限速步骤，因为一旦产生回文双着丝粒染色体，它将不可避免地进入坏螺旋（BFB循环）并导致特定基因组区域的积累（回文基因扩增）。 在这个建议中，我们将定义顺式（基因组）和反式（遗传）的作用因素，是重要的启动回文基因扩增在人类肿瘤。我们以前已经表明，在哺乳动物细胞模型中，DNA反向重复（DNA-IR）预先存在于基因组中，与相邻的DSB，极大地促进回文基因扩增。因此，基因扩增的第一步是DSB启动的，DNA-IR重复序列之间的非法重组。这导致了我们的DNA水平模型; DNA-IR内的折叠回（链内）退火将产生一个具有发夹帽末端的染色体，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：人类基因组中预先存在的DNA-IR是顺式作用（基因组）决定因素（Aim 1），而处理发夹帽末端的基因和诱导DSB的生理条件是重要的反式作用决定因素（Aim 2）。为了实现我们的目标，我们建立了独特的实验系统，可以克服研究回文DNA的困难。基因组方法采用了一种新的技术，富集回文DNA，并确定重要的DNA-IR的回文基因扩增在原发性人类肿瘤。我们的细胞培养系统被设计成测量在具有各种遗传背景的细胞中在DNA-IR处发生的DSB启动的非法重组。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中结构变异的影响，多态性DNA-IR可能是个体对基因扩增易感性的重要预测因素。 公共卫生相关性： 我们研究的主要目标是确定在人类癌症中产生基因扩增的机制。基因扩增是指癌细胞中基因的额外拷贝的积累，通常会导致细胞异常生长和肿瘤的侵袭行为。因此，我们对潜在机制的鉴定将更好地了解癌症的进展，并最终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.