Single molecule detection of DNA replication errors

DNA复制错误的单分子检测

• 批准号: BB/W006014/1
• 负责人: Conrad Nieduszynski
• 金额: $ 55.8万
• 依托单位: Earlham Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW006014%2F1
• 关键词: Single; molecule; detection; DNA; replication

All cells contain a complete copy of the organism's DNA, the genetic blueprint of life, packaged into discrete units called chromosomes. Since new cells need a copy of the genetic material, the chromosomes must be completely and accurately replicated before the cell can divide. Our research aims to determine how cells ensure that the replication of each chromosome is completed accurately. Problems can occur when the machinery that copies the DNA encounters an obstacle. This can cause the DNA replication machinery to slow or pause which in turn can give rise to duplications, the expansion/contraction of repeated sequences or even lead to breaks in both strands of the DNA. Therefore, although obstacles rarely cause a problem for DNA replication, when they do the consequences can be catastrophic for the cell.Rare events, such as pausing of the replication machinery, can be difficult to detect, since most DNA replication is occurring normally. These rare, but serious events, present a 'needle in a haystack' problem for researchers. We have developed a high-throughput DNA sequencing technology that allows us to study the kinetics of DNA replication in vivo on single molecules. This technology allows us to rapidly search for the 'needle in the haystack' and identify rare, but serious, events such as the slowing down or pausing of the DNA replication machinery. We will apply this approach to determine what DNA sequences can pose an obstacle to the DNA replication machinery; what protein factors assist in getting past such obstacles; and how pausing of the replication machinery is linked to errors during the copying process. This is important because a single DNA replication error on one chromosome in a single cell division can give rise to genomic disorders, including cancer.

所有细胞都包含生物体 DNA（生命的遗传蓝图）的完整副本，被包装成称为染色体的离散单元。由于新细胞需要遗传物质的副本，因此在细胞分裂之前染色体必须完整且准确地复制。我们的研究旨在确定细胞如何确保每条染色体的复制准确完成。当复制 DNA 的机器遇到障碍时，就会出现问题。这可能会导致 DNA 复制机制减慢或暂停，进而导致重复、重复序列的扩张/收缩，甚至导致 DNA 两条链断裂。因此，虽然障碍很少会导致 DNA 复制出现问题，但一旦出现，后果可能对细胞造成灾难性的后果。罕见事件（例如复制机制暂停）可能很难检测到，因为大多数 DNA 复制都是正常发生的。这些罕见但严重的事件给研究人员带来了“大海捞针”的问题。我们开发了一种高通量 DNA 测序技术，使我们能够研究单分子体内 DNA 复制的动力学。这项技术使我们能够快速搜索“大海捞针”，并识别罕见但严重的事件，例如 DNA 复制机制减慢或暂停。我们将应用这种方法来确定哪些 DNA 序列会对 DNA 复制机制构成障碍；哪些蛋白质因子有助于克服这些障碍？以及复制机制的暂停如何与复制过程中的错误相关联。这一点很重要，因为单次细胞分裂中一条染色体上的单个 DNA 复制错误可能会导致基因组疾病，包括癌症。