Tag: Diana Libuda

Defining the roles of conserved DNA repair complexes in maintenance of C. elegans meiotic genome integrity

Presenter(s): Alina Salagean—Biology

Faculty Mentor(s): Erik Toraason, Diana Libuda

Session 3: The Substance of Us

Most organisms utilize meiosis, a specialized form of cell division, to produce reproductive cells such as sperm and eggs . Failure to maintain genomic integrity during meiosis can result in serious diseases, including infertility and cancer . The Structural Maintenance of Chromosomes 5/6 complex (SMC-5/6), its E3 SUMO ligase subunit NSE-2, and the BRCA1/BARD1 heterodimer are conserved protein complexes implicated in ensuring accurate meiotic DNA repair and are known to genetically interact . However, the specific mechanisms by which these proteins interact to preserve genome integrity is unknown . To determine the NSE-2 specific and NSE-2 independent meiotic functions of the SMC-5/6 complex in meiotic DSB repair, we utilized immunofluorescence imaging and a mortal germline phenotype assay to assess smc-5 and nse-2 C . elegans mutants . Our findings suggest a separation of function within the SMC-5/6 complex, which performs NSE-2 dependent functions promoting efficient meiotic DSB repair and NSE-2 independent functions in preservation of germline immortality . Finally, to define epistatic relationships between BRC-1/BRD-1, SMC-5/6, and NSE-2 in DNA repair, we assessed the germline sensitivity to exogenous DNA damage by scoring the brood viability of pairwise brc-1, smc-5, and nse-2 double mutants . These data reveal that exogenous DNA damage repair is differentially regulated within meiotic prophase I and implicate SMC-5/6 as a central regulator of both NSE-2 and BRC-1 dependent DSB repair . Taken together, our research defines fundamental genetic mechanisms and interactions preserving genomic integrity .

Defining the roles of conserved DNA repair complexes in maintenance of C. elegans meiotic genome integrity

Presenter(s): Alina Salagean—Biology

Faculty Mentor(s): Erik Toraason, Diana Libuda

Session 6: Interact & React

Most organisms utilize meiosis, a specialized form of cell division, to produce reproductive cells such as sperm and eggs . Failure to maintain genomic integrity during meiosis can result in serious diseases, including infertility and cancer . The Structural Maintenance of Chromosomes 5/6 complex (SMC-5/6), its E3 SUMO ligase subunit NSE-2, and the BRCA1/BARD1 heterodimer are conserved protein complexes implicated in ensuring accurate meiotic DNA repair and are known to genetically interact . However, the specific mechanisms by which these proteins interact to preserve genome integrity is unknown . To determine the NSE-2 specific and NSE-2 independent meiotic functions of the SMC- 5/6 complex in meiotic DSB repair, we utilized immunofluorescence imaging and a mortal germline phenotype assay to assess smc-5 and nse-2 C . elegans mutants . Our findings suggest a separation of function within the SMC-5/6 complex, which performs NSE-2 dependent functions promoting efficient meiotic DSB repair and NSE-2 independent functions in preservation of germline immortality . Finally, to define epistatic relationships between BRC-1/BRD-1, SMC-5/6, and NSE-2 in DNA repair, we assessed the germline sensitivity to exogenous DNA damage by scoring the brood viability of pairwise brc-1, smc-5, and nse-2 double mutants . These data reveal that exogenous DNA damage repair is differentially regulated within meiotic prophase I and implicate SMC-5/6 as a central regulator of both NSE-2 and BRC-1 dependent DSB repair . Taken together, our research defines fundamental genetic mechanisms and interactions preserving genomic integrity .

Development of a new live imaging technique to uncover the mechanisms of heat- induced male infertility

Presenter(s): Cailan Feingold—Biology

Faculty Mentor(s): Diana Libuda, Cori Cahoon

Session 3: The Substance of Us

Male infertility affects approximately one-third of couples who are unable to conceive . Exposing mammalian spermatogenesis to elevated temperatures causes 40% of primary male infertility cases; however, the mechanisms behind this heat-induced male-specific infertility are largely unknown . Similar to mammals, Caenorhabditis elegans also display heat-induced sperm-specific infertility . Following heat-stress, C . elegans spermatocytes have increases in DNA damage that correlate with a premature loss of chromosome structures essential for meiotic chromosome segregation . Using live imaging, I will examine the dynamic relationship between this heat-induced DNA-damage and disassembly of meiotic chromosome structures in spermatocytes . To circumvent immobilization issues with existing current live imaging techniques, I am developing and implementing a new conditional, immobilization method for live imaging fluorescently tagged proteins in both sexes of intact worms . This novel method utilizes the auxin-inducible degron system, which targets degradation of degron tagged proteins in the presence of auxin, and thus can be used to specifically degrade genes that cause severe paralysis . Based on the gene location and predicted function, I selected three genes to degron tag (unc-104, unc-52, unc-18) . Using fertility assays, I confirmed that loss of these three gene products does not interfere with meiosis or fertility . Overall, this novel live imaging system will allow for conditional paralysis of living worms during live imaging experiments, enabling us to examine the dynamics of the heat-induced defects during spermatogenesis .