Isolation and characterization of genes involved in DNA damage response and telomere maintenance in Caenorhabditis elegans Public Deposited

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Last Modified
  • March 21, 2019
Creator
  • Boerckel, Julie Ann
    • Affiliation: College of Arts and Sciences, Department of Biology
Abstract
  • Many cells, including somatic cells, are mortal, they divide a set number of times and then senesce or undergo cell death. However, germline and cancer cells have the ability to divide indefinitely and are thus considered immortal. In an attempt to better understand how cellular immortality is maintained I have been studying the pathways needed to maintain germline immortality in the nematode C. elegans. Several genes in various genome stability pathways have been identified to be exclusively activated in both cancer and germ cells suggesting they are important for cellular immortality. One pathway is the length maintenance of telomeres, the ends of chromosomes. Previously, members of the Rad9/Rad1/Hus1 DNA damage response complex were identified in C. elegans as being essential not only for germline immortality but also telomere maintenance. Based on these findings, two independent forward genetic screens were conducted to identify other genes involved in DNA damage response and telomere maintenance by first selecting for mutants that are hypersensitive to ionizing radiation (which causes double-strand DNA breaks) or resistant to hydroxyurea (which triggers S-phase cell cycle arrest) and then for the Mortal Germline phenotype, sterility after propagation for multiple generations. One mutant allele, yp4, was isolated as being resistant to hydroxyurea and is highly defective for cell cycle arrest in response to both ionizing radiation and hydroxyurea. Further characterization of yp4 revealed that it is required for germline immortality and displays phenotypes indicative of progressive telomere shortening. Additionally, a reverse genetic screen identified hpr-17, which encodes a Rad17 homolog, as being the RFC clamp loader that facilitates the Rad9/Rad1/Hus1 complex activity in response to DNA damage and in telomerase-mediated telomere replication. Aside from the two genes identified to be involved in telomere maintenance, 15 radiation hypersensitive mutants were isolated that carry mutations in genes that are essential for germline immortality at 25°C, whereas 32 hydroxyurea resistant mutants were isolated that are essential for germline immortality at all temperatures. Further analysis of DNA damage response pathways revealed the mutants isolated represent a number of genes that play roles in cell cycle arrest and/or apoptosis. These mortal germline mutants suggest that at least two DNA damage response pathways, independent of telomerase, are required for maintenance of germ cell immortality.
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  • In Copyright
Advisor
  • Ahmed, Shawn
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Open access
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