During DNA replication, forks often stall upon encountering obstacles blocking their progression. Cells will act to speedily remove or overcome such barriers, thus allowing complete synthesis of chromosomes. This is the case for R-loops, DNA/RNA hybrids that arise during transcription. One mechanism to remove such R-loops involve DNA/RNA helicases. Here, I have shown that one such helicase, Sen1, associates with replisome components during S phase in the model organism S. cerevisiae. I demonstrate that the N-terminal domain of Sen1 is both sufficient and necessary for the interaction of the protein with the replisome. I also identified Ctf4 as one of at least two replisome interactors of Sen1. By mutational analysis, a mutant of Sen1 (Sen1-3) that cannot interact with the replisome was created. This mutant is healthy on its own but is lethal in the absence of both RNase H1 and H2. Overexpression of the sen1-3 allele from the constitutive ACT1 promoter is able to suppress this synthetic lethality, suggesting that Sen1 travels with replisomes in order to be quickly recruited at sites of R-loops that impair fork progression so as to remove those R-loops. In some cases, cells exploit fork stalling for biologically important processes. This is the case in Sz. pombe, where an imprint prevents complete DNA replication, triggering cell-type switching. This imprint is dependent on Pol1, a component of the replisome. Importantly, a single imprinting-defective allele of pol1 has been identified to date. Using in vitro assays, I have shown that this Pol1 mutant has reduced affinity for its substrates and is a correspondingly poor polymerase. By generating novel alleles of pol1, I have also demonstrated that switching-deficiency correlates with the affinity of Pol1 for its substrates in vivo. Finally, two interactors of Pol1 (Mcl1Ctf4 and Spp1Pri1 ) have been shown to have switching defects. S. cerevisiae and Sz. pombe have similar yet distinct genetic nomenclature conventions. Given that both model organisms were used in this study, it is important to highlight the conventions for both organisms to prevent confusion. In S. cerevisiae, wildtype gene names are expressed as a three letter, uppercase and italic name followed by a number (e.g. SEN1). The three letter name often corresponds to the screen through which the gene in question was originally identified. Mutants are generally designated with the same three letter but in lower case (unless the mutant is dominant) and with an allele designation (e.g. sen1∆, sen1-1 and sen1-2). Because of historical context, the allele designations vary in format (e.g. leu2-3,112 is a mutant of LEU2). Protein names are given as a three letter name with the first letter in uppercase (e.g. Sen1). This is also true for mutant proteins, with the added allele designation (e.g Sen1-1 and Sen1-2). In this study, I have generated constructs of the SEN1 gene and these constructs are referred to as SEN1 (X-Y), where X and Y refer to the first and last residues being encoded for. The corresponding proteins are referred to as Sen1 (X-Y). Different promoters have been used and, where appropriate, the promoters are expressed similarly to their wildtype gene names (e.g. GAL1, SEN1 and ACT1). In Sz. pombe, wildtype gene names are expressed as a three letter, lowercase and italic name followed by a number (e.g. pol1). Mutants are generally designated in the same format but with an allele designation. Like in S. cerevisiae, the allele designation varies widely (e.g. pol1-1, pol1-H4 and pol1-ts13). Additionally, because of the historical context, some (but not all) alleles of pol1 are referred to as swi7 to reflect the fact that they are defective for cell-type switching. Similar to the situation in S. cerevisiae, proteins names are given as a three letter name with the first letter in uppercase for both wildtype and mutants (e.g. Pol1 and Swi7-1). Sometimes, for the sake of comparison, genes or proteins are referred to their S. cerevisiae orthologues (e.g. swi1TOF1 and Swi1Tof1 , respectively). Several protein tags have been used in this study. When written in gene form, they were written in capital letters and italicized, irrespective of the host (e.g. 5FLAG) and when in protein form, they were written in capital, irrespective of the host (e.g. 5FLAG).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:737755 |
| Date | January 2017 |
| Creators | Appanah, Rowin |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/100501/ |
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