Investigating the molecular mechanisms of meiotic recombination

Gray, Stephen (2013) Investigating the molecular mechanisms of meiotic recombination. Doctoral thesis (PhD), University of Sussex.

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Abstract

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein Spo11. During DSB formation, Spo11 becomes covalently attached to the 5’ DSB ends. Removal of Spo11 is essential to repair the DSB by homologous recombination. Spo11 is removed endonucleolytically creating short-lived Spo11-oligonucleotide products. Here I demonstrate that:

1. Spo11-oligonucleotide products are not detected in recombination mutants believed to be defective in meiotic DSB formation.
2. When DSB repair is delayed, Spo11-oligonucleotides persist for longer.
3. Processing of Spo11-DSB ends to create Spo11-oligonucleotides is largely dependent on Mec1 and Tel1 activity.

In the process of investigating Spo11-oligonucleotide degradation, it was observed that a mutant defective in both the meiotic recombination checkpoint and in DSB repair failed to accumulate the expected level of DSBs. Work described here leads to the proposal of a DSB feedback mechanism that functions though the Mec1 (ATR) pathway to increase the efficiency of DSB formation. By contrast, Tel1 (ATM) functions to inhibit DSB formation, agreeing with recently published data. However, the data presented also suggests that Tel1 acts alongside the Mec1 pathway to promote DSB formation. It is therefore proposed that such positive and negative regulation creates a homeostatic mechanism to ensure that an optimum frequency of DSBs is formed.

In wildtype cells, single –stranded DNA resection relies only on the Exo1 nuclease. In checkpoint defective cells resection length is increased. Results described here demonstrate that in a checkpoint defective strain, resection functions through Exo1, Sgs1/Dna2 and a third currently untested resection mechanism, likely to be Mre11 dependent.

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: 13 Mar 2013 14:04
Last Modified: 12 Oct 2015 11:36
URI: http://sro.sussex.ac.uk/id/eprint/43900

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