The role of 5'-tyrosyl-DNA-phosphodiesterase two (TDPT-1) mediated suppression of DNA topoisomerase 2 (TOP-2) during meiosis in C. elegans
Date
2023
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Publisher
University of Delaware
Abstract
Meiosis, a specialized cell division involving a single cycle of DNA replication followed by two cycles of chromosome segregation, is a highly coordinated event. Chromosome segregation errors during meiosis have hazardous consequences such as birth defects, genetic diseases, infertility and malignancy. DNA Topoisomerase II (Topo II) is required to relieve the topological stress associated with the unwinding of DNA during replication, recombination and sister chromatid segregation. In addition, Topo II has been also found to maintain chromosome structure. The focus of Topo II studies has been on its roles during mitosis but its role in meiosis is not very well defined. Our lab previously characterized an allele of top-2 [top-2(it7)] in Caenorhabditis elegans with a male meiosis-specific phenotype. top-2(it7) is a temperature sensitive (ts) allele and top-2(it7ts) sperm that develop at the restrictive temperature of 24°C have chromosome segregation defects at anaphase I of meiosis, which results in embryonic lethality after fertilization. ☐ In chapter 2, employing EMS mutagenesis followed by a genetic suppressor screen, I identified putative genes that interact with top-2 during meiosis. I used a combination of RNAi and CRISPR/Cas9 genome editing to validate the responsible suppressor mutations identified by whole-genome sequencing. I also examined the meiotic chromosome segregation in the suppressors and found that chromosome segregation defects are ameliorated in the suppressors. Interestingly, seven of the 11 identified suppressors that were identified had point mutations in the same gene, 5'-tyrosyl-DNA phosphodiesterase two (TDP2, C. elegans tdpt-1). Human TDP2 is involved in the removal of trapped TOP2-DNA protein complexes (TOP2-DPCs). ☐ tdpt-1 mutation-mediated suppression of the top-2 mutant phenotype poses an important question: What is the specific role of TDPT-1 in relation to TOP-2 in meiosis and how do these proteins interplay to help in accurate chromosome segregation during male meiosis? In chapter 3, I utilized genetic analysis to elucidate the role of these tdpt-1 mutations in context of top-2(it7) during meiosis. I found that tdpt-1 mutant mediated suppression is due to loss of function of the protein and also that the tdpt-1 mutations do not have a phenotype independently of top-2(it7) in meiosis. In addition, I found that fewer double stranded breaks (DSBs) are generated in the top-2(it7) mutant at 24°C but a tdpt-1 suppressor mutation restores DSBs levels. Moreover, I characterized and validated the functionality of endogenous fluorescent tag at N-terminus of TDPT-1 protein and utilized this tool to show that TDPT-1 is expressed in nuclei of the germ line and the soma of C. elegans. Chapter 4 is the extension of chapter 3 where I utilized an E. coli system to purify and biochemically characterize the properties of the TDPT-1 mutant proteins in relation to wild type. I showed that the suppressing mutations in TDPT-1 impair the phosphodiesterase activity and affect the thermal stability of TDPT-1. ☐ Together, the results from my dissertation work suggest the regulation of meiotic chromosomes through an interplay of two special enzymes whose function in meiosis have not been well defined previously. This dissertation has laid a foundation for more work required to further illuminate how exactly these enzymes function for accurate meiotic progression.
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Keywords
Birth defects, Genetic diseases, Chromosome segregation, Meiotic progression, Caenorhabditis elegans