Studying DNA replication dynamics in Schizosaccharomyces pombe using next generation sequencing

Regions at which replication is initiated are called origins of replication and the fidelity of DNA replication is crucial to genome stability. A number of factors can affect the progression of DNA replication and origin firing. In unperturbed cells, origins are fired in a fixed temporal manner, a phenomenon known as the DNA replication timing program. In this thesis, a novel next generation deep sequencing technique was optimised to study the progression of DNA replication through repetitive and heterochromatic regions in a variety of genetic backgrounds. The loss of Rif1, a protein implicated in a number of processes, such as facultative heterochromatin formation, DNA damage response and DNA replication timing control, yielded the most unexpected results. In addition to local effects in origin firing activity around Rif1 binding sites, rif1? resulted in a complete loss of the global replication timing program. Based on these data, this thesis further explores the relationship between the global replication timing program and the landscape of origin firing efficiencies. In metazoans, the establishment of the DNA replication timing program was linked to the nuclear distribution of chromatin. Here, we describe the role that the tethering of chromatin to the nuclear periphery plays in establishing the global replication timing program in S. pombe. Finally, we present a model explaining the global replication timing program in S. pombe and the role that global origin firing plays within it.