Investigating initial DNA unwinding during budding yeast replication

Abstract

DNA unwinding is a central, but poorly understood process that facilitates the establishment of bi-directional replication. Sustained DNA unwinding is carried out by the replicative helicase. Most helicases require some single-stranded or untwisted DNA in order to function, and it is this initial DNA unwinding that this study aimed to characterise. There are two possible timepoints when DNA unwinding could occur: during loading of the helicase onto DNA or during the activation of that helicase, as the replisome forms. During this project each possible timepoint for initial unwinding was investigated in chronological order in vitro and S phase was scanned for the appearance of DNA unwinding in vivo. Techniques for in vitro DNaseI, nuclease P1 and potassium permanganate footprinting using supercoiled plasmid DNA were established within the first part of this project. These were implemented to reveal that no DNA unwinding occurs during helicase loading, but on completion of loading origin DNA is distorted. The latter part of this study comprised two methods for investigating DNA unwinding during helicase activation. The first method employed an in vitro approach to reconstitute the process of helicase activation during replisome formation. Here, purified proteins were used to supplement S phase yeast extracts. These mixtures were incubated with helicase pre-loaded onto origin DNA and the resulting complexes analysed using Western blotting. A novel in vivo footprinting approach, designed to access potentially unstable unwound DNA within the yeast nucleus was also executed. A single-stranded DNA-specific nuclease was expressed within yeast to target unwound DNA. DNA was extracted throughout S phase and analysed by primer extension. This study highlighted many of the obstacles associated with characterising DNA unwinding. Nevertheless, it reports the development of in vitro and in vivo assays for DNA unwinding. In addition, several new reagents were produced that will benefit future studies.