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Title: The role of Arabidopsis thaliana mismatch repair proteins in meiotic recombination
Author: Blackwell, Alexander Robert
ISNI:       0000 0004 8508 0897
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Meiosis is a conserved eukaryotic cell division that increases genetic diversity in progeny. During meiosis, homologous chromosomes pair and undergo reciprocal exchange, called crossover. Meiotic recombination initiates from DNA double strand breaks, which are repaired using either sister or homologous chromatids as templates. When meiosis occurs in heterozygous (hybrid) organisms, interactions between homologous chromosomes have the potential to generate mismatched DNA structures. Several proteins with roles in DNA mismatch repair (MMR) are known to influence meiotic recombination in several eukaryotes. In Arabidopsis thaliana this includes three MSH heterodimers (MSH2-MSH3, MSH2-MSH6 and MSH2-MSH7) that recognise mismatched nucleotides and have demonstrated roles in repressing meiotic crossovers in hybrid plants. To further investigate the meiotic function of MMR genome-wide, I generated a series of msh2 mutant introgression lines in three different genetic backgrounds – Ct-1, Ler-0 and CLC – which have varying patterns and levels of polymorphism. Consistent with the function of MSH2 as a hybrid-specific anti-recombinase, I observed significant crossover increases in the chromosome arms in all three hybrid msh2 mutants. However, I also found evidence for accession and region specific effects of msh2 on crossover frequency. For example, crossovers appeared to decrease over centromere proximal regions in msh2 compared to wild type. A genotyping-by-sequencing experiment was performed to generate genome-wide crossover maps in two Arabidopsis hybrids, with and without MSH2 function. This revealed that total crossover number remained unchanged in the MMR-deficient hybrids, whilst crossovers redistributed into regions of reduced polymorphism density. This relationship was counter to my expectation that MMR would repress crossovers most strongly in divergent regions. However, this relationship was observed across varying physical scales, from 1–100 kilobases. This confirms a positive relationship between polymorphism and meiotic crossovers in Arabidopsis hybrids, and reveals a novel role for MSH2 in mediating this effect. In addition to the investigation of MSH2 in meiotic recombination, I present an analysis of the genome-wide distribution of MutS homolog 4 (MSH4). MSH4, and its binding partner MutS homolog 5, evolved from their ancestral role in MMR and now function exclusively to promote meiotic crossovers in the ZMM pathway. I present a genome-wide chromatin-immunoprecipitation-sequencing analysis of the binding profile of MSH4, and analyse its distribution at varying scales. This has revealed novel relationships between MSH4, the meiotic cohesin subunit REC8, and features of the chromatin and recombination landscapes. Together, these results advance our understanding of meiotic recombination in plants, and raise further questions about the regulation of crossovers in eukaryotes more broadly.
Supervisor: Henderson, Ian R. Sponsor: Gatsby Charitable Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Meiosis ; crossover ; recombination ; genetic diversity ; polymorphism ; mismatch ; MMR ; MSH2 ; MSH4 ; ZMM ; Arabidopsis ; ChIP-seq