Genome wide identification of priming sites for Okazaki fragments

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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.