Graduate Thesis Or Dissertation
 

Characterization of Neurospora crassa and Fusarium graminearum mutants defective in repeat-induced point mutation

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  • Mutation of repetitive DNA by repeat-induced point mutation (RIP) is a process that occurs in many filamentous fungi of the Ascomycota during the sexual cycle. Concurrently, direct DNA repeats are often deleted by homologous recombination at high frequency during the sexual cycle. Thus, the processes of RIP and deletion compete to either mutate or remove repetitive DNA from the genome of filamentous fungi during sexual cycles. Both processes contribute to genome streamlining by controlling proliferation of transposable elements and by limiting expansion of gene families. While the genetic requirements for deletion by homologous recombination are well known, the mechanism behind the specific detection and mutation of repetitive DNA by RIP has yet to be elucidated as only a single gene essential for RIP, rid, has been identified. We have developed Fusarium graminearum as a model organism for the study of RIP by showing that it mutates repetitive DNA frequently during the sexual cycle and that the mutations due to RIP are dependent on rid. Further, we have sequenced a genetic mapping strain of F. graminearum (00-676-2) and identified 62,310 single nucleotide polymorphisms (SNPs) compared to the reference strain (PH-1). The SNP map will be useful for quickly mapping new mutants by bulk segregant analysis and high-throughput sequencing for which bioinformatic tools were specifically developed. The groundwork has thus been laid for identification of novel RIP mutants in F. graminearum, which being homothallic has a major advantage for identification of recessive mutations. We used a forward genetics approach to shed light on the mechanism of RIP in Neurospora crassa. Two rrr mutants that dominantly r̲educe R̲IP and r̲ecombination were characterized and identified as different mutated alleles of the same gene, rrr-1[superscript L496P] and rrr-1[superscript G325N] by bulk segregant analysis and high-throughput sequencing. Bioinformatic characterization suggests RRR-1 belongs to a previously uncharacterized group of dynamin-like proteins, which are generally involved in membrane fission and fusion. RRR-1-GFP localizes to the nuclear membrane, but not DNA, suggesting it affects RIP and recombination frequency indirectly by altering nuclear membrane dynamics during sexual development and thereby altering temporal aspects of RIP and recombination. We used a reverse genetics approach to determine whether high frequency RIP and homologous recombination of repetitive DNA during the sexual cycle are linked mechanistically or spatio-temporally. We tested strains where genes important for deletion by homologous recombination were knocked out and found all to be completely RIP competent except mre11, which, while sterile in homozygous deletion crosses, displayed lower RIP frequency in heterozygous crosses. This suggests that mre11 has roles in homologous recombination as well as non-homologous end joining may be important for RIP. Collectively, this work developed methods for efficiently mapping mutations and identified a novel protein that reduces RIP and recombination frequency but did not identify any mechanistic link between the two processes.
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