Tag: Oral Session 3 M

Chemoreceptor Zinc-Binding Protein Domains Sense Hypochlorous Acid

Presenter(s): Dan Tudorica

Faculty Mentor(s): Arden Perkins

Oral Session 3 M

The Chemoreceptor Zinc-Binding domain (CZB) is a protein module common in host-associated bacteria that seems to regulate bacterial chemoreceptors that control motility. The ligand these protein domains sense remains uncertain, however CZB domains contain a cysteine that binds to zinc, a chemical moiety that is known to be reactive with bleach (HOCl). Thus, my hypothesis is that CZBs are responsible for sensing HOCl, which is a prevalent antibacterial agent synthesized by human neutrophils to combat infections. Using the fluorescence of a sample of purified CZB, my data indicate the protein’s structure changes in response to physiologically-relevant concentrations of HOCl, consistent with a mechanism for signal transduction. Furthermore, by examining the circular dichroism spectrum of CZB under increasing concentrations of HOCl, I identified this structural change as a loss of alpha-helicity.

I also examined the hardiness of CZB-possessing bacteria in vivo in the presence of varying concentrations of HOCl. I found that the bacterial pathogens Salmonella and Helicobacter pylori, which possess CZB-regulated chemoreceptors, can tolerate acute treatments of HOCl and remain motile, and were more resistant than Escherichia coli, which has a CZB-regulated diguanylate cyclase but lacks a CZB-regulated chemoreceptor. E. coli, however, proved to be more tolerant of surviving high levels of HOCl over 6-12 hours. In summary, my research suggests CZB domains have the surprising capability to sense HOCl, the strongest oxidant generated by the human immune system, and that bacteria that colonize humans may use these sensors for different purposes in their colonization strategies.

Effects of Alisertib in Acute Lymphoblastic Leukemia NSG Mouse Models

Presenter(s): Corinne Togiai

Faculty Mentor(s): Bill Chang

Oral Session 3 M

Acute Lymphoblastic Leukemia (ALL) is a common cause for disease-related mortality in children and adolescents. As we have made great strides in curing ALL we have identified subsets of diseases that continue to have a poor prognosis. To develop novel targeted therapies in hopes to advance the treatment of these diseases, our lab initiated the use of rapid, state-of-the-art genetic and functional assays to identify aberrant activated pathways from primary patient leukemic samples. Results through collaborative research with the Knight Cancer Institute Leukemia Research Group, have identified significant hypersensitivity to different cellular pathway inhibition. Our current proposal builds on these findings. We have identified a unique hypersensitivity of certain subsets of leukemic cells to inhibition of the Aurora class of cell cycle kinases. Aurora kinases are members of serine/threonine kinases that play pivotal roles for the cell to faithfully undergo mitosis. Studies have shown that certain cancers are heavily dependent on the activity of these kinases beyond mitosis and that these kinases can be targeted by specific small molecule drugs. Our preliminary data is the first to identify subsets of ALL that are hypersensitive to aurora kinase inhibition. What remains unknown is the mechanism of hypersensitivity in subsets of ALL as well as in vivo validation.

Other future directions in parallel aim towards determining the mechanism of hypersensitivity to Aurora kinase inhibitors in subsets of ALL, and developing in vivo models testing single agent and combination therapy specifically targeting these pathways.

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.

Piwi-piRNA pathway protein PRG-1 represses in temperature-induced DNA damage in spermatocytes

Presenter(s): Fountane Chan

Faculty Mentor(s): Diana Libuda & Nicole Kurhanewicz

Oral Session 3 M

Poster 47

Session: Sciences

Half of infertility cases worldwide involve male-factor subfertility. As awareness and frequency of male infertility has grown, it is increasingly important to understand the underlying mechanisms of these major human health concerns. Developing sperm are particularly sensitive to fluctuations in temperature, requiring a narrow isotherm of 2-7°C below core body temperature. Although both oocytes and spermatocytes undergo meiosis, the specialized form of cell division that produces haploid sex cells, elevated gonadal temperatures have been shown both to impair only male fertility and produce excess DNA damage specifically in spermatocytes. Preliminary work using the powerful roundworm model Caenorhabditis elegans suggests the Piwi-piRNA pathway, a highly conserved genome maintenance pathway, is involved in temperature-induced DNA damage. Absence of worm-specific Argonaute proteins (WAGO), primary effector proteins of the Piwi pathway, results in considerably elevated DNA damage upon heat-shock. Interestingly, a panel of mutants deficient in the C. elegans Piwi protein, PRG-1, which functions upstream of WAGOs, demonstrate highly variable degrees of heat-induced DNA damage. This variability is likely due to acquired mutations stemming from inadequate germline surveillance over multiple generations. To circumvent this issue by controlling the number of generations a strain is without PRG-1, we generated a conditional knockdown mutant of PRG-1. Utilizing this mutant, we found that in the absence of PRG-1 after one generation, spermatocytes demonstrate exacerbated levels of heat-induced DNA damage, similar to WAGO null mutants. Taken together, my data suggest a key role for PRG-1 and male-specific components of the Piwi pathway in heat-induced DNA damage in spermatocytes.