Genome wide identification of priming sites for Okazaki fragments

我们的遗传蓝图是由染色体分子编码的，染色体分子由DNA和染色质组成。染色质决定了单个基因的表达状态。在我们的一生中，细胞会受到损伤和死亡。因此，组织通过细胞分裂不断再生。每次细胞分裂时，DNA和相关的染色质都必须复制。这是通过DNA复制过程发生的。DNA复制始于基因组中称为起源的特定位点，并从这些位点以双向方式进行。由于DNA由两条反平行的链组成，并且这两条链只能在一个方向上复制，因此其中一条链，即前导链，以连续的方式合成，而另一条链，即滞后链，则作为片段合成，随后将它们组合在一起形成最终产物。每次DNA复制在后链上启动时，就会发生引物事件，但实际上，是什么决定了这些引物被放在哪里，我们一无所知。我们已经开发了一种新的方法来确定启动位点的位置在基因组水平上使用下一代测序。利用这项技术突破，我们将回答有关复制过程的基本问题，这些问题以前是不可能回答的。我们将能够识别基因组中复制过程减慢或终止的位置，因为在这些位置上启动是位点特异性的。我们还将能够识别在复制过程中由于不频繁或过于频繁的启动事件而出现“问题”的区域。最后，我们将研究染色质是否会影响引物的放置位置。该项目将回答有关所有细胞生命背后的复制过程的一些非常基本的问题，并将增加我们对这一基本过程中可能出现问题的了解，从而使我们深入了解癌症和衰老背后的遗传不稳定性。

Our genetic blueprint is encoded by chromosomal molecules that consist of DNA as well as chromatin. The chromatin determines expression states of individual genes. As we go through life, cells get damaged and die. Thus, tissues are constantly regenerating through cell divisions. Every time a cell divides, the DNA and the associated chromatin have to be duplicated. This happens by the process of DNA replication. DNA replication is initiated at specific sites in the genome called origins, and progresses in a bidirectional manner from these. Since DNA consists of two anti-parallel strands, and these two strands only can be copied in one direction, one of the strands, the leading, is synthesized in a continuous manner, while the other, the lagging, is synthesized as fragments that are subsequently put together to form the final product. Each time the DNA replication is initiated on the lagging strand, a priming event takes place, but virtually nothing is known about what determines where these primers are put down. We have developed a novel method for determining the position of the priming sites on the genomic level using Next Generation Sequencing. Using this technical breakthrough, we will answer fundamental questions about the replication process that would not have been possible to answer before. We will be able to identify positions in the genome where the replication process slows down or terminates, as at these positions priming is site-specific. We also will be able to identify region were there are "problems" during the replication process due to infrequent or too frequent priming events. Finally, we will investigate whether chromatin affects where primers are put down. The project will answer some very fundamental questions about the replication process underlying all cellular life, and will increase our knowledge about what can go wrong during this essential process, thus giving us insight into the genetic instability that underlies cancer and ageing.