Developing single molecule methods to study DNA replication dynamics in yeasts

To ensure the faithful transmission of genetic information during the cell division cycle, the entire genome must be accurately and completely duplicated prior to segregation of the identical sister chromatids. Perturbation of this process can have severe consequences for a cell, potentially resulting in cell death or genomic instability defects that can contribute to the development of disease. Therefore, it is important to understand how such events are avoided during normal genome replication and the impact of both endogenous and exogenous stresses on the process. For decades, various experimental techniques have been developed and applied to gain insight into the dynamics of genome replication, each contributing to an ever- growing knowledge base on the subject. More recent advances have included the increasing progression towards single cell and single molecule approaches to expand the understanding of cell-to-cell heterogeneity in how replication proceeds. The current work aimed to contribute to these efforts by developing a novel approach for the analysis of single molecule replication dynamics. It was postulated that detecting DNA polymerase usage or lagging strand synthesis by optical mapping in ultra-long single molecules would enable the evaluation of each molecule’s pattern of replication. A protocol for mapping Okazaki fragments was established and tested by Bionano Genomics Saphyr analysis. Preliminary results supported the detection of labelled Okazaki fragments, showing potential for further protocol and analytical optimisation to validate this new method. Furthermore, the experimental procedure for the application of an existing single molecule technique in a different model organism was explored. Nanopore sequencing-based detection of nucleotide analogue incorporation during replication was tested in Schizosaccharomyces pombe. Initial analyses indicated that the experimental system was not fully optimised for the approach and required alterations to enable the inference of replication dynamics information from long sequencing reads.