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

 描述（由申请人提供）：我们研究的目的是开发一种新方法，用于纯化和表征具有不寻常结构的新型癌症特异性DNA，这些DNA来源于DNA复制控制缺陷的基因组区域。遗传物质的精确复制对保持基因组的完整性至关重要。为了精确地完成复制，DNA复制受到严格的控制：（1）在单个细胞周期中复制起点仅发射一次，并且（）当复制叉停滞或崩溃时重新启动/修复复制叉。复制控制中的缺陷可能 导致计划外的复制启动（重新复制）或不完整的复制， 叉子卡住了这些事件是基因组不稳定性的主要原因，基因组不稳定性是肿瘤增殖和转移的主要特征。因此，准确测量和定位复制控制缺陷区域的方法有助于我们理解基因组不稳定性的机制。重新复制和复制叉停滞都可能导致非生产性复制，尽管对这种非生产性复制的命运知之甚少。我们假设，非生产性复制可能会导致产生DNA与不寻常的结构。与我们的假设一致，我们在癌细胞中天然存在的叉停滞位点鉴定了这样的DNA。这一发现促使我们提出了一种从癌细胞中纯化这种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不寻常结构的DNA来源的基因组位置，从而确定DNA复制存在问题的位置。这些信息是新颖的，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纯化这种DNA的方法。目的1利用分子生物学方法建立一种从癌细胞中富集具有异常结构DNA的方法。目标2将使用NGS来确定在天然存在的复制叉停滞位点处的DNA富集。目标3将鉴定来自复制起点的具有不寻常结构的DNA，这些DNA正在进行再复制。我们在全基因组范围内调查癌症特异性不寻常DNA结构的经验支持了拟议研究的可行性。这种技术的成功发展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.