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

描述（由申请人提供）：我们的研究目标是明确癌细胞中基因扩增的启动机制。确定起始机制应该提供重要的知识，因为扩增常常驱动肿瘤进展和治疗抵抗。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-融合桥循环，BFB循环）是潜在的启动机制；(1)染色体断裂导致断裂端在复制后融合（姐妹染色单体融合），产生具有两个着丝粒的新生回文（反向复制）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端部断裂（break）；(3)断端进入另一个循环。因此，初始步骤是一个潜在的限速步骤，因为一旦产生回文双中心染色体，它将不可避免地进入一个坏螺旋（BFB循环），并导致特定基因组区域的积累（回文基因扩增）。在本提案中，我们将定义顺式（基因组）和反式（遗传）的作用因素，这些因素在人类肿瘤中启动回文基因扩增中很重要。我们之前在哺乳动物细胞模型中表明，基因组中存在的DNA倒置重复序列（DNA- ir）与相邻的DSB极大地促进了回文基因扩增。因此，基因扩增的第一步是dsb发起的，DNA-IR重复之间的非法重组。这就引出了我们的dna水平模型；DNA- ir内部的折叠（链内）退火会产生一条末端带有发夹帽的染色体，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：人类基因组中存在的DNA-IRs是顺式作用（基因组）决定因素（Aim 1），而处理发夹端和诱导dsb的生理条件的基因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。基因组学方法采用了一种新的技术来富集回文DNA，并在原发性人类肿瘤中鉴定出重要的回文基因扩增DNA- irs。我们的细胞培养系统旨在测量具有各种遗传背景的细胞中dsb引发的DNA-IR非法重组的发生。这些实验应该共同确定特定的DNA-IRs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-IRs可能是个体对基因扩增易感性的重要预测因子。

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.