Browsing by Author "Kwah, Ji Kent"
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Item Mutations in tyrosyl-DNA phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during Caenorhabditis elegans spermatogenesis(Journal of Biological Chemistry, 2024-06-28) Kwah, Ji Kent; Bhandari, Nirajan; Rourke, Christine; Gassaway, Gabriella; Jaramillo-Lambert, AimeeMeiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5′-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.Item Regulation of f237 filamentous prophage excision and replication(University of Delaware, 2020) Kwah, Ji KentVibrio parahaemolyticus is a seafood-borne enteric pathogen found in marine environments, associated with fish and shellfish. A genetic marker for V. parahaemolyticus pandemic strains is the presence of a filamentous prophage f237 (also known as VfO3K6). The f237 prophage integrates site specifically via recombination into the dif site (attB site) on chromosome 1. f237 reproduces through episomal replication after excision from the chromosome. In this thesis, we examined the role of quorum sensing (QS) regulators on the replication and excision of f237 prophage. Quorum sensing is a bacterial regulatory system that allows cell to cell signaling via small molecules known as autoinducers. The level of autoinducer correlates with changes in cell density that result in a signaling cascade that activates and deactivate LuxO, the QS response regulator. In a ΔluxO mutant, the high cell density master regulator OpaR is constitutively expressed. In this mutant, most f237 genes were significantly upregulated. We performed PCR assays to examine the presence of the attachment sites of both integrated (attB) and excised (attP) forms of f237 prophage in wild type, luxO and opaR mutants. This analysis showed that the levels of attB and attP of f237 increased under high cell density conditions when OpaR is present. Whereas, we observed a reduction is attB and attP levels in the opaR deletion mutant suggesting it is a positive regulator of f237 excision and replication. We examined the binding of OpaR to the regulatory regions of f237 and showed binding in the promoter region of ORF10 and ORF1. Lastly, we showed that ORF1, the homolog of RstA, a phage replication protein, is required for the replication of f237. This is the first instance of direct regulation of a filamentous phage by a host quorum-sensing regulator.