Single molecule detection of DNA replication errors

所有细胞都含有生物体DNA的完整副本，这是生命的基因蓝图，被包装成称为染色体的离散单元。因为新的细胞需要遗传物质的副本，所以在细胞分裂之前，染色体必须完全和准确地复制。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，可能会出现问题。这会导致DNA复制机制减慢或暂停，进而导致复制、重复序列的扩展/收缩，甚至导致DNA的两条链断裂。因此，尽管障碍很少对DNA复制造成问题，但一旦发生，后果可能对细胞造成灾难性的影响。罕见的事件，如复制机制的暂停，可能很难检测到，因为大多数DNA复制都是正常进行的。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，使我们能够研究在体内单分子上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，如DNA复制机制的减速或暂停。我们将应用这一方法来确定哪些DNA序列会对DNA复制机制构成障碍；哪些蛋白质因素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联系。这一点很重要，因为在一次细胞分裂中，一条染色体上的一个DNA复制错误就会导致包括癌症在内的基因组疾病。

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.