Tag: Erik Toraason

Investigating the Role Of Genomic Positioning in Directing Meiotic Double-Strand DNA Break Repair

Presenter(s): Rachel David − Biochemistry

Faculty Mentor(s): Diana Libuda, Erik Toraason

Oral Session 3M

Research Area: Biological Sciences

Funding: UO Alden Scholar Research Award, NICHD Grant

During meiosis, the specialized form of cell division that produces gametes, cells utilize recombination to maintain genomic integrity and promote proper chromosome segregation, ensuring fertility. Double-strand DNA breaks (DSBs), which serve as substrates for homologous recombination, are intentionally induced during meiosis. A fraction of DSBs must be repaired
as crossover recombination events with the homologous chromosome to forge a physical connection required to facilitate proper chromosome segregation. Although DSBs are induced across the genome, crossovers in C. elegans are preferentially formed along chromosome arms and not the center of chromosomes. What determines this crossover preference along chromosome arms is not well known. Intriguingly, previous studies in C. elegans have indicated that the crossover landscape is not determined by chromatin marks or specific sequence motifs. However, proximity of a DSB to the synaptonemal complex (SC), a meiosis-specific proteinaceous structure that connects homologous chromosomes together, has been suggested to influence DSB repair outcomes. To determine how genomic positioning of a DSB affects its repair outcome, I am exploiting genetic assays developed by the Libuda lab that enable controlled induction of a single DSB at a known genomic location and assess how that induced DSB was repaired. Utilizing CRISPR/Cas9 genome editing, I have targeted these assays to four unique loci that differ in position both along the chromosome length and in proximity to the SC. Together, our studies will elucidate how the position of a DSB within the genome influences how it is repaired to maintain genome integrity.

SMC-5/6 Facilitates Efficient DSB Repair During Meiosis in C. elegans

Presenter(s): Cordell Clark − Biology

Faculty Mentor(s): Diana Libuda, Erik Toraason

Poster 73

Oral Session 3M

Research Area: Biological Science

Funding: OURS Program Summer 2017, Dr. Diana Libuda’s laboratory is funded in part by an NICHD grant

Sexually reproducing organisms depend upon meiosis to form haploid sex cells necessary for reproduction. Despite the inherent risks of DNA damage to genome integrity, meiotic cells intentionally induce double strand DNA breaks (DSBs) throughout the genome. A specific and limited number of DSBs must be repaired as crossovers with the homologous chromosome to promote proper chromosome segregation. DSBs are induced in excess of the permitted number of crossovers. DSBs not repaired as crossovers must be repaired to maintain genomic integrity. In the Libuda lab, we have designed an assay to determine the repair outcome of a single induced DSB. We have demonstrated that in addition to the homologous chromosome, the sister chromatid is used as a repair template during DSB repair in C. elegans meiosis. My research aims to uncover the mechanisms that facilitate intersister repair, which are currently unknown. Experiments in multiple species have demonstrated that SUMOylation is required for crossover formation. Our preliminary immunofluorescence experiments in SUMO deficient mutants reveal SUMOylation is required for DSB repair in C. elegans oocytes. Prior research has hypothesized that the SMC5/6 complex, which contains a SUMO ligase subunit (NSE-2), facilitates intersister recombination. Using our intersister repair assay, I have demonstrated that the SMC5/6 complex is required for efficient intersister repair and intersister crossovers. My ongoing experiments are directly testing whether the SMC5/6 complex promotes intersister recombination via the NSE-2 SUMO ligase subunit. Overall, our studies are defining the mechanisms that facilitate intersister recombination to ensure genome integrity during sperm and egg development.

SMC-5/6 E3 SUMO ligase subunit NSE-2 is required for robust repair of meiotic DNA double-strand breaks

Presenter(s): Alina Salagean

Faculty Mentor(s): Diana Libuda & Erik Toraason

Oral Session 3 M

Most organisms utilize meiosis, a specialized form of cell division, to produce haploid gametes such as sperm and eggs. Failure to maintain genomic integrity during meiosis can cause infertility and cancer. Using the model organism Caenorhabditis elegans, previous work has demonstrated that the conserved Structural Maintenance of Chromosomes 5/6 complex (SMC-5/6) is required for robust repair of double-strand DNA breaks (DSBs) in late meiotic prophase I. The specific mechanisms by which SMC-5/6 promotes DSB repair remain unknown. One subunit of the SMC- 5/6 complex, the E3 SUMO ligase NSE-2, has been implicated in DNA repair in multiple organisms. To identify the specific contributions of NSE-2 to meiotic DSB repair and fertility, we generated four nse-2 null mutants using CRISPR/Cas9 genome editing and assessed their phenotypes associated with genome integrity across generations. Utilizing these nse-2 mutants, we find that similarly to SMC-5, NSE-2 is required for a germ line-response to exogenous DNA damage. In contrast, unlike SMC-5, NSE-2 is not required for maintenance of fertility over generations. These data suggest NSE-2 is required for either a specific subset of functions of the SMC-5/6 complex or the efficient function of SMC-5/6. Our future experiments will utilize both genetic assays and immunofluorescence imaging techniques to distinguish between these hypotheses. Taken together, our research defines mechanisms preserving genomic integrity and fertility across generations.

Mechanisms of sister chromatid repair during meiotic double-strand DNA break repair

Presenter(s): Anna Horacek

Faculty Mentor(s): Diana Libuda & Erik Toraason

Oral Session 3 M 

Poster 68

Session: Sciences

Most sexually reproducing organisms utilize meiosis, a specialized form of cell division, to generate haploid gametes such as eggs and sperm. Meiotic cells utilize recombination to repair double-strand DNA breaks (DSBs) with either the sister chromatid or homologous chromosome as a repair template. Although recombination with the homologous chromosome has been extensively studied, little is known about engagement of the sister chromatid during meiotic DSB repair. To characterize sister chromatid recombination, we have developed a sister chromatid repair (SCR) assay in Caenorhabditis elegans. The SCR assay contains engineered nucleotide polymorphisms enabling the detection of gene conversions between sister chromatids, which arise from the nonreciprocal exchange of sequences between chromosome templates and indicate recombination intermediates. Analysis of these conversions tracts indicate that throughout meiotic prophase I: 1) sister chromatid repair intermediates remain central to the site of DSB induction; and, 2) sister chromatid repair is highly processive, as template switching is not observed. Interestingly, the length of conversion tracts, indicating the extent of DSB resectioning, changes in the presence of a homolog repair template. In the absence of a homolog repair template, the conversion tract size is uniform throughout prophase I. When a homolog repair template is present, large conversion tracts (>210bp) are observed only in late prophase I, suggesting the presence of the homolog repair template may affect the extent of DSB resectioning in late prophase I. Taken together, our work presents a comprehensive analysis of meiotic sister chromatid recombination and defines mechanisms fundamental to the preservation of 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 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 .