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Title: The role of histone modification in DNA double-strand break repair in Schizosaccharomyces pombe
Author: Deegan, Rachel Sarah
ISNI:       0000 0004 2738 469X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2012
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DNA double-strand breaks (DSBs) are highly genotoxic lesions, which when incorrectly repaired can lead to gross chromosomal rearrangements and tumourigenesis through oncogene activation or loss of heterozygosity at tumour suppressor loci. Chromatin structure and therefore histone modification can play a key role in the repair of these lesions through affecting the access of repair machinery to the break. To identify novel histone-modifying enzymes involved in DSB repair, nineteen histone-modifying mutants from an S. pombe deletion library were screened for altered DSB repair. Eleven genes were identified as required for normal DSB repair: set1⁺, set2⁺, mst2⁺, sir2⁺, set3⁺, set11⁺, hat1⁺, set13⁺, clr4⁺, gcn5⁺ and elp3⁺. Two genes, hat1⁺ and set13⁺ are required for efficient homologous recombination (HR). In addition, four genes set1⁺, set2⁺, set3⁺ and mst2⁺ are required for non-homologous end-joining (NHEJ). Analysis of set1Δ, set2Δ, set3Δ and mst2Δ deletion mutants showed impaired NHEJ was associated with significantly increased levels of repair by HR, supporting the existence of a competitive relationship between these mechanisms of repair. Further work focused on Set2, a H3K36 methyltransferase. In addition to a role for Set2 in NHEJ, a role for Set2 in promoting efficient DNA replication was identified. This is likely through regulation of MBF-dependent genes. However, this altered progression of DNA replication was not responsible for the repair phenotype seen. The methyltransferase activity of Set2 was required for its role in NHEJ and DNA replication. Further analysis of a H3K36R mutant identified an additional role for H3K36 in DSB repair. Results indicate that H3K36 is also acetylated by Gcn5; quantitative analysis of DSB repair in a gcn5Δ mutant, identified a role for Gcn5 in promoting HR repair. Modification of H3K36 by methylation and acetylation is mutually exclusive, and preliminary analysis of the levels of these modifications suggests that they are cell cycle controlled. The findings presented here support a model whereby differential modification of H3K36 by Gcn5 and Set2 defines the DNA repair mechanism utilised. Methylation of H3K36 by Set2, which peaks in G1, promotes NHEJ whereas acetylation of H3K36 by Gcn5, which peaks in S phase, promotes HR.
Supervisor: Humphrey, Timothy Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biology (medical sciences) ; Biology (medical sciences) ; DNA repair