Thesis summary Sample Clauses

Thesis summary. The work presented in this thesis attempts to shed some light on the mechanisms of Okazaki fragment maturation, termination, and chromatin replication using the model organism S. cerevisiae. Work in recent years has led to considerable progress in our understanding of the early stages of replication, including initiation and elongation. However, significantly less is known about the events that occur at end of replication, as well as the mechanisms through which replication occurs on chromatin. This is mostly due to difficulties studying these processes in vivo. The complete reconstitution of these processes with purified proteins in vitro will provide a powerful tool for studying how these complicated processes work in vivo. To that end, I adopted a biochemical approach and built upon the previously reported reconstitution of replication initiation and elongation with purified proteins from S. cerevisiae (Xxxxxx et al. 2015; Xxxxxx et al. 2017). Initially, I developed purification protocols for the proteins involved in Okazaki fragment maturation and identified the minimal set of proteins required for both complete lagging strand synthesis and termination in vitro (Chapter 3). Interestingly, I obtained evidence showing that excess loaded Mcm2-7 double-hexamers are inhibitory to replication on chromatin, and identified a novel pathway for their removal involving Pif1 (Chapter 4). Later, I developed expression strains and purification protocols for the proteins implicated in de novo chromatin assembly: CAF-1, Asf1, Rtt106, Vps75, and Rtt109. I then identified the minimal set of proteins required for this process in vitro: histones, CAF-1 and Asf1 (Chapter 5). Finally, I determined the requirements for Okazaki fragment maturation and termination on chromatin, and established a fully-reconstituted system for studying complete chromatin replication, including both parental histone recycling and de novo chromatin assembly, in vitro (Chapter 6). In addition to providing a powerful tool for future studies of these processes in vitro, this work provides significant insights into replication and chromatin assembly. To our knowledge, this is the first time Pif1 has been implicated in removing loaded Mcm2-7 from DNA. Indeed, it was previously unknown how (or when) excess Mcm2-7 loaded during G1 are removed from DNA. Furthermore, this is the first reported reconstitution of de novo chromatin assembly in its entirety with purified proteins, and the first study to ident...