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Title: Analysis of nucleotide synthesis and homologous recombination repair in Schizosaccharomyces pombe
Author: Blaikley, Elizabeth Jane
ISNI:       0000 0004 5355 2239
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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Nucleotide synthesis is a conserved and highly regulated response to DNA damage, required for the efficient repair of DNA double strand breaks (DSB) by homologous recombination (HR). This is essential to prevent loss of heterozygosity (LOH) and maintain genome stability. The aim of this study was to identify new genes important for HR through roles in damage-induced nucleotide synthesis. A screen was performed to identify S. pombe gene deletion strains whose DSB sensitivity was suppressed by deleting the ribonucleotide reductase (RNR) inhibitor spd1+ to promote nucleotide synthesis. The screen identified a number of genes including ddb1+, cdt2+, rad3+ and csn1+ which have known roles in nucleotide synthesis. Distinct roles were identified for the DNA damage checkpoint in suppressing LOH. rad3+, rad26+, rad17+ and the rad9+, rad1+ and hus1+ genes encoding the 9-1-1 complex were required for DNA damage-induced nucleotide synthesis through Cdt2 induction to promote Spd1 degradation. The HR repair defect of rad3+ and rad26+ deletion strains was partially suppressed by spd1+ deletion. However, the HR repair defect of rad17+, rad9+, rad1+ and hus1+ deletion strains was not suppressed. An additional role was confirmed for Rad17 and the 9-1-1 complex in preventing LOH by promoting DSB resection. A role was identified for the Gcn5 histone acetyl transferase (HAT) protein module, consisting of Gcn5, Ngg1, Ada2 and Sgf29, in suppressing DSB sensitivity by promoting nucleotide synthesis. This was independent of Cdt2 or RNR protein levels. The Gcn5 HAT module was also found to regulate DSB repair pathway choice consistent with previous observations. Deletion of gcn5+, ngg1+ or ada2+ decreased HR and increased non-homologous end joining. Surprisingly, deletion of spd1+ in a gcn5∆, ngg1∆ or ada2∆ background also promoted HR. This predicts a role for nucleotide pools in regulating DSB repair pathway choice. Eleven other candidates showed repeatable suppression of DSB sensitivity following spd1+ deletion. However many of these candidates did not show reduced nucleotide levels. This suggests deleting spd1+ may also suppress DSB sensitivity by a different mechanism.
Supervisor: Humphrey, Timothy Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Oncology ; DNA damage signalling ; DNA double strand break ; homologous recombination ; Schizosaccharomyces pombe