Browsing by Subject "Mismatch repair"
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Item Open Access Roles of RNA interference and DNA mismatch repair in maintaining genomic integrity in Cryptococcus pathogens(2022) Priest, Shelby JordanMicroorganisms must regulate genomic stability to strike a balance between excessive deleterious mutation and evolutionary stagnation to successfully compete and endure within their ecological niches. Two important mechanisms involved in maintaining genomic stability are RNA interference (RNAi) and DNA mismatch repair (MMR). RNAi defends the host genome by targeting double-stranded viral RNAs and aberrant endogenous RNAs for degradation. Endogenous sources of aberrant RNAs include transcripts derived from transposable elements and repetitive sequences as well as transcripts with inefficiently spliced introns. Transcription and translation of these endogenous aberrant RNAs is often considered deleterious to the host because transposable elements are capable of replicating and spreading throughout the genome, a process that can disrupt genes and destabilize chromosomes. The DNA MMR pathway canonically detects mismatches caused by DNA damage or errors during DNA replication. After recognizing mismatches, MMR pathway components recruit the appropriate proteins for removal and repair of the mismatched nucleotide. In addition to this role, MMR pathway components are also involved in the rejection of homeologous, or only partially homologous, meiotic recombination intermediates. This activity mediates a critical role in the maintenance of species boundaries, by inhibiting successful recombination between the genomes of two sufficiently divergent organisms, often preventing the production of viable or fertile progeny.The first chapter of this dissertation begins by introducing pathogenic Cryptococcus species, their ability to mediate disease in humans, and various aspects of their genomes and life cycles. Following the introduction to Cryptococcus, factors known to mediate genomic instability in fungi are described. In Chapter 2, the identification and characterization of two clinical Cryptococcus neoformans isolates with significantly increased mutation rates due to RNAi loss and rampant mobilization of a transposable element are detailed. Chapter 3 describes the impact of loss of a functional MMR pathway on the species boundary between C. neoformans and Cryptococcus deneoformans, sister species within the pathogenic Cryptococcus species complex. In Chapter 4, experimental procedures for conducting genetic crosses with Cryptococcus, isolating meiotic products, and many factors impacting these methods are presented. The conclusions of each preceding chapter are then summarized in Chapter 5, where I also put forth further questions and directions for each project. In Appendices A and B, two ongoing projects focused on the identification of additional RNAi-deficient C. neoformans strains as well as work to discover novel RNAi components are respectively described. Lastly, Appendices C and D include supplementary tables from Chapters 2 and 3, respectively.
Item Open Access Transcription Factors as Competitors in Gene Regulation and DNA Damage Repair(2022) Zhang, YuningTranscription factors (TFs) bind genomic DNA to regulate gene expression. In the cell, the genome is decorated with numerous proteins, including nucleosomes and proteins involved in processes such as DNA repair and replication, which could compete with TFs. While the competition with nucleosomes is well studied, TFs can also compete with other DNA-binding proteins (e.g. other TFs, DNA repair enzymes, polymerases). The rules and the impact of such competition remain largely unknown. Here, we investigate how TFs compete with each other and with repair enzymes, and we reveal the significant role TFs play as competitors in multiple pathways.To capture the binding profiles of competing TFs, we designed a quantitative cell-free assay that we applied to study Cbf1-Pho4 competition in yeast and MYC-MAD competition in human. We found that TFs greatly influence each other’s occupancy, in a way that is dictated by the proteins’ divergence in DNA-binding specificity. Analyses of ChIP-seq data confirmed that the patterns of TF-TF competition, as observed in vitro, are preserved in the nuclear environment. Furthermore, gene expression data suggests that Cbf1-Pho4 competition plays a critical role in the specific activation of target genes in the cell. In the MYC-MAD system, we found that quantitative in vitro knowledge facilitates the interpretation of in vivo ChIP-seq data and reveals subtle signals in gene regulatory networks, demonstrating the advantage of combining in vitro quantification with in vivo detection. Next, we adapted our assay to study the competition between TFs and DNA repair enzymes. Recently (Afek et al. 2020) we showed that TFs bind with high affinity to mismatches, which can result from replication errors. We thus hypothesized that TFs can compete with TDG, the glycosylase that recognize T-G mismatch and initiates base excision repair, and MutS, the mismatch-binding enzyme that initiates mismatch repair. Our high-throughput competition assay showed that, as predicted, the binding of both repair enzymes to DNA decreases significantly in the presence of TFs. In addition, the magnitude of the decreases in repair enzyme binding correlates well with the TF binding levels, indicating specific competition. This suggests that, in the cell, TFs bound to mismatches may affect repair and lead to increased mutagenesis at regulatory sites. Overall, our study proposes an approach for studying competition between DNA-binding proteins in a quantitative and high-throughput manner, and highlights the significance of this competition not only for gene regulation (where TFs are known to play an important role), but also in DNA repair.