Small circular DNA as a signature of defects in DNA replication control

 描述(申请人提供)：我们的研究目标是开发一种新的方法来纯化和表征新的癌症特异性DNA，这些DNA具有来自DNA复制控制缺陷的基因组区域的不寻常结构。准确复制遗传物质对于保持基因组的完整性是必不可少的。为了准确地完成复制，DNA复制受到严格的控制：(1)在单个细胞周期中只触发一次复制起点，以及()在叉子停滞或坍塌时重新启动/修复复制叉子。复制控制中的缺陷可能 导致计划外复制启动(重新复制)或复制不完整 卡壳的叉子。这些事件是基因组不稳定的主要原因，而基因组不稳定是肿瘤增殖和转移的主要特征。因此，一种准确测量和绘制复制控制缺陷区域的方法有助于我们理解基因组不稳定的机制。重新复制和复制分叉停滞都可能导致非生产性复制，尽管人们对这种非生产性复制的命运知之甚少。我们假设，非生产性复制可能导致产生具有不寻常结构的DNA。与我们的假设一致，我们在癌细胞中自然发生的分叉失速位置发现了这样的DNA。这一发现促使我们提出了一种从癌细胞中提纯这种DNA的方法。通过使用下一代测序(NGS)来表征DNA，我们可以系统地确定具有异常结构的DNA的基因组位置，从而确定DNA复制存在问题的位置。这些信息是新的，并促进了我们对癌细胞复制控制缺陷的理解。我们将通过追求三个目标来开发一种纯化这种DNA的方法。目的1将利用分子生物学方法建立一种从癌细胞中富含具有特殊结构的DNA的方法。AIM 2将使用NGS来确定自然发生的复制分叉停滞位置的DNA浓缩情况。AIM 3将鉴定具有不寻常结构的DNA，这些DNA来自经历了重新复制的复制起始点。我们在全基因组中研究癌症特异性异常DNA结构的经验支持了拟议研究的可行性。这种技术的成功开发将使我们能够在大规模研究中进一步研究具有不寻常结构的DNA。

 DESCRIPTION (provided by applicant): The goal of our study is to develop a new method for purifying and characterizing novel cancer specific DNA with unusual structures that derive from genomic regions with defects in DNA replication control. Accurate duplication of genetic material is essential to maintain genome integrity. To precisely complete the duplication, DNA replication is under strict controls: (1) firing of replication origins only once in a single cell cycle, and () restart/repair replication forks when forks stall or collapse. Defects in replication control could result in either unscheduled replication initiation (re-replication) or incomplete replication with stalled forks. These events are a major cause of genome instability, a major enabling characteristic of tumor proliferation and metastasis. Therefore, an approach that accurately measure and map the region suffering from defects of replication control promotes our understanding of mechanisms underlying genome instability. Both re-replication and replication fork stalling could result in unproductive replication, although little is known about the fate of such unproductive replication. We hypothesize that unproductive replication could result in producing DNAs with unusual structures. Consistent with our hypothesis, we identified such DNAs at a naturally occurring fork stalling site in cancer cells. This finding led us to propose a method that purifies such DNA from cancer cells. By characterizing the DNA using Next Generation Sequencing (NGS), we can systematically determine the genomic locations where the DNAs with unusual structures derive from and thus where DNA replication has problems. Such information is novel and promotes our understanding of defects in replication control in cancer cells. We will develop a method for purifying such DNA by pursuing three aims. Aim 1 will use molecular biology approach to establish a method for enriching DNAs with unusual structures from cancer cells. Aim 2 will use NGS to determine the enrichment of DNA at naturally occurring replication fork stalling sites. Aim 3 will identify DNA with unusual structure derived from replication origins undergoing re-replication. Our experiences in investigating cancer-specific unusual DNA structures in genome-wide support the feasibility of the proposed studies. Successful development of such technology would allow us to investigate the DNA with unusual structures further in large scale studies.