Investigating double-strand break formation and repair in meiosis

Johnson, Dominic (2017) Investigating double-strand break formation and repair in meiosis. Doctoral thesis (PhD), University of Sussex.

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Meiotic recombination is a complex process that requires tight regulation to ensure accurate chromosomal segregation and to prevent DNA double-strand breaks (DSBs), introduced to initiate meiotic recombination, from becoming damaging. Spo11 introduces DSBs via a topoisomerase-like reaction during meiosis. In this thesis I present work investigating the mechanisms that regulate the formation and repair of the protein-linked DSBs created by Spo11 and topoisomerase II (Top2). Initiation of Spo11-DSB resection is conducted by the Mre11-Rad50-Xrs2 (MRX) complex and Sae2 protein, which nucleolytically removes Spo11 covalently bound to oligonucleotides via a phosphotyrosine bond. Sae2 activity is controlled by post-translational modifications and regulation of its oligomeric state. Here I present data characterising the phenotype of Sae2 proteins mutated at putative Mec1/Tel1 phosphorylation sites (Chapter 3). The human TDP2 protein, hydrolytically removes proteolysed topoisomerase II (Top2) from the 5ʹ end of Top2-DSBs. Here I show that TDP2 is also active upon the phosphotyrosine bond between Spo11 and DNA in vitro (Chapter 4). Removal of Spo11 from the 5ʹ′end of doublestranded DNA by TDP2 permits resection by lambda exonuclease but no resection is observed by the primary meiotic 5ʹ to 3ʹ exonuclease, Exo1, in vitro. This suggests an evolutionary benefit of Spo11-DSB processing by the MRX complex and Sae2 at generating a substrate that permits Exo1 resection instead of the hydrolytic Spo11 removal mechanism by TDP2 (Chapter 4). Utilising TDP2 activity, I have developed a novel method to map Spo11-DSBs genome-wide with single nucleotide resolution (Chapter 5). The spatial patterning of meiotic DSBs is controlled in yeast by the ATM/ATR homologs Tel1/Mec1. Results from this new genome-wide DSB mapping method suggest that the kinase activity of Tel1 regulates hyper-local repression of coincident Spo11-DSBs (Chapter 6). v Utilising TDP2 and the nucleotide resolution mapping procedure for Spo11, Saccharomyces cerevisiae topoisomerase II (Top2) was also mapped genome-wide, which indicated that there are preferential sites for Top2 cleavage in vivo (Chapter 7). In the future this procedure can be adapted to map other protein-DNA complexes in vivo in a wide range of organisms. Collectively the work presented in this thesis further elucidates the mechanisms underpinning the spatial patterning of Spo11-DSBs in meiosis, the subsequent repair of meiotic DSBs, and also contributes to our understanding of the location of Top2 cleavage sites in vivo.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 08 Jun 2017 14:40
Last Modified: 08 Jul 2019 10:46

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