Analyzing the structural and functional changes of TOP-2 variants in Caenorhabditis elegans

Date
2019
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University of Delaware
Abstract
During meiosis, cells undergo one round of DNA replication followed by two rounds of cell division to form haploid gametes. In meiosis I, homologous chromosomes segregate while in meiosis II, the sister chromatids segregate before cytokinesis to form haploid cells. Topoisomerase II is an enzyme that helps to facilitate DNA disentanglements. This enzyme has been studied extensively in mitosis but not in meiosis. Previously, we demonstrated that a temperature sensitive mutant, top-2(it7), causes chromosomes to fail to segregate in spermatogenesis when incubated at 24°C. The top-2(it7) mutation changes an arginine at amino acid 828 to cysteine. This mutation lies within an α/β fold of TOP-2 called the tower subdomain that is located within the catalytic domain, a region that has been proposed to interact with DNA. Structural modeling of the TOP-2 protein indicates that the wild-type arginine makes several interesting contacts with other amino acids, including a salt bridge with glutamate at amino acid site 960. We hypothesize that the arginine at position 828 is particularly important for the structure and function of the enzyme. I have characterized several amino acid substitutions at Arg828. I found that an Arg828Ala change has high embryonic viability at 15°C but significantly reduced viability when incubated at 25°C. This mutation has intermediate segregation defects at 25°C compared to top-2(it7). An Arg828Trp change results in sterility due to the absence of germline development. Characterization of an Arg828Lys change reveals reduced viability when incubated at 24°C that is exacerbated at 25°C. Examination of the germline shows chromosomal segregation defects that are not as penetrant as those seen in top-2(it7). Future directions will include the examination of protein levels in the various top-2 mutants, the characterization of additional amino acid substitutions, and assays to determine the effects of the mutations on TOP-2 enzymatic activity.
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