RPA complexes and tool development for the study of DSB repair in the Caenorhabditis elegans germline
Year: 2021
Type: dissertation
Abstract: Meiosis is a specialized cell division required for gamete formation in diploid eukaryotic organisms. This process halves the amount of genomic content and involves two reductional divisions where in meiosis I homologs are segregated, and sister chromatids are divided in meiosis II. In most organisms, proper segregation of homologous chromosomes in meiosis I requires the formation and repair of programmed meiotic double-strand DNA breaks (DSBs) that will allow for stable pairing of homologs prior the meiotic anaphase I. DSBs generated during meiosis require repair by homologous recombination (HR) to form essential crossovers that provide the physical tether between homologous chromosomes. After formation of meiotic DSBs, broken ends are processed by specialized machinery to produce single-stranded DNA (ssDNA) which will engage in homology search and repair by meiotic HR. One of the first proteins to respond to ssDNA is the protein complex named replication protein A (RPA). This complex recognizes and preferentially binds ssDNA to prevent the formation of secondary structures and allow for recruitment of subsequent proteins that will facilitate HR. In most organisms, RPA is a heterotrimeric complex composed of a large, medium, and small subunit (Rpa1, Rpa2, and Rpa3 respectively), where each subunit is critical to the function of the complex. RPA subunit composition in C. elegans differs from other organisms in that there are two copies of the medium subunit of RPA (RPA-2 and RPA-4), and there is a lack of the canonical small subunit (RPA-3). Here we show that RPA-1 is sufficient for somatic replication, unlike other organisms, and we show that RPA-4 performs a novel function in which it attenuates the loading of the recombinase RAD-51. Furthermore, we show that RPA-4 marks sites of replication defect DNA damage and promotes apoptosis of marked nuclei. Taken together, these data indicate that RPA subunits in C. elegans are unique in composition and function.This work also shows the development of tools useful in studying meiosis in C. elegans. Using genome editing technologies (CRISPR and MosSCI) we have utilized the split-sfGFP technology for the efficient tagging of target proteins (including RPA-1). We use CRISP to tag proteins with a small amino acid sequence (GFP11) that when complimented by the large subunit (GFP1-10) will fluoresce. Germline-specific fluorescence was accomplished by regulating expression of GFP1-10 with germline promoter and 3’UTR. Using this technology, we can observe the localization of target proteins within the germline in vivo. Furthermore, this system is useful for tagging proteins which may otherwise be difficult to tag with full-length GFP. We also demonstrate the development of a heat-shock inducible system designed to generate targeted DSBs in the germline. Our results show that we were able to induce the expression of a wrmScarlet tagged I-SceI in the germline following heat-shock. However, it is unclear if the germline expressed I-SceI can generate DSBs in the germline. This body of work demonstrates some of genome editing tools available for use in C. elegans germline and the methods in which to regulate expression within this tissue.
DOI: 10.17077/etd.005786
Authors: Adam Hefel, Andrew Forbes, Deborah Dawson, Marc Wold, Tina Tootle, Sarit Smolikove, Anna Malkova
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4231009801