Genomic single molecule analysis of chromatin structure around DNA replication origins in Saccharomyces cerevisiae

Origin licensing initiates DNA replication by loading proteins on to DNA. In eukaryotes origin licensing varies among inactive replication origins and can be analysed by looking at chromatin structure. This has predominantly been studied using population-based approaches including MNase-seq. These mask the heterogeneity in interactions between DNA-binding events that occur on the same DNA molecule. In this thesis the development of a single molecule genomic footprinting approach will be discussed, with a focus on identifying an appropriate marker of accessible DNA. Firstly, two chemicals, 4,5’,8-trimethylpsoralen (TMP) and angelicin, were assessed using polyacry- lamide denaturing gels and mass spectrometry, which showed that TMP could modify DNA while angelicin modification of DNA was not observed. Subsequently, nanopore sequencing did not detect the TMP modification. Secondly, DNA methylation was investigated which involved validating the sequence specificity of M.EcoGII, a non-specific adenine methyltrans- ferase, and developing a Droplet Digital PCR assay to quantitatively assess methylation levels at individual loci. Finally, methylation was used in combination with nanopore sequencing to analyse chromatin structure in Saccharomyces cerevisiae through both in vitro chromatin preparations and novel in vivo inducible Dam and M.EcoGII systems. These showed that S. cerevisiae tolerates expression of an adenine methyltransferase, methylation in a variety of sequence contexts is detectable by nanopore sequencing and some chromatin structure can be observed however further optimisation is required.