Abstract
Eukaryotic DNA replication machinery disrupts the structure of chromatin as the replication fork progresses through DNA. Importantly, the histone content must be duplicated every cell cycle in addition to the DNA. Whilst a number of insights into the initiation and elongation phases of replication on chromatin have been revealed from decades of work in vivo and in vitro, many mechanistic details of the later stages of replication remain poorly understood. In this study, I used an in vitro biochemical approach to build upon the work described by Yeeles et al. (2017) and Kurat et al. (2017). I reconstituted Okazaki fragment maturation and identified the minimal set of proteins involved in this process on both naked and chromatinised DNA: Fen1, DNA ligase I, PCNA, and DNA polymerase d. I then showed that these proteins – in addition to those required for replication initiation – are sufficient to generate relaxed, covalently closed daughter DNA.The process of termination is slow and inefficient relative to the rest of replication, however, and leads to the accumulation of late replication intermediates. I showed that Pif1 is able to enhance termination by (1) decreasing the amount of time it takes to occur and (2) resolving the late replication intermediates generated in the absence of Pif1. Furthermore, I found that excess Mcm2-7 are inhibitory to replication on chromatin and must be removed from the DNA. I then identified a novel pathway for Mcm2-7 removal during replication involving Pif1. It has been known for many years that cells licence more origins of replication than are used in each cell cycle. In fact, many species (including humans) have been shown to load many more Mcm2-7 than there are origins on DNA. This is the first indication that they need to be removed during replication, and the data presented in this thesis provides a mechanism for how they are removed from the DNA. 4
Subsequently, I reconstituted replication-coupled chromatin assembly with purified proteins in vitro for the first time and identified the minimal set of proteins required for this process: histones, CAF-1, and Asf1. I also showed that regular spacing of nucleosomes on nascent DNA requires a nucleosome remodeller such as ISW1a. I then established a system for studying complete chromatin replication in vitro, including both the recycling of parental histones and de novo chromatin assembly by CAF-1 and Asf1.
| Date of Award | 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Shaun Thomas (Supervisor) |