Functional studies of accessory factors associated with base excision repair Public Deposited

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  • March 21, 2019
Creator
  • Pachkowski, Brian Francis
    • Affiliation: Gillings School of Global Public Health, Department of Environmental Sciences and Engineering
Abstract
  • Exposure to environmental and cellular mutagens is ubiquitous and, as a consequence, DNA is constantly faced with the possibility of becoming damaged. Base excision repair (BER) removes some of this damage to limit the impact of these exposures on cell physiology and ultimately human health. The function of core BER enzymes may be enhanced by other protein accessory factors, namely poly(ADP-ribose) polymerase-1 (PARP-1) and x-ray repair cross complementing gene 1 (XRCC1). The main hypothesis of this research was that genetic approaches using cellular knockout and complementation models can evaluate whether the accessory proteins PARP-1 and XRCC1 are determinants of BER efficiency. While numerous biochemical studies have implicated PARP-1 in BER, the role of this protein in BER is somewhat uncertain. The first aim of this research was to evaluate the role of PARP-1 in BER in vertebrate cells. Chicken cells lacking PARP-1 were treated with an alkylating agent under different scenarios with subsequent endpoint measurements. PARP-1 was necessary as a survival factor during chronic exposure but did not appear relevant in acute exposures until the late stages of BER. In the absence of exposure, the DNA lesions measured were equal between PARP-1 proficient and deficient cells. XRCC1 acts as a scaffold for numerous protein interactions necessary for proficient BER. However, the presence of polymorphic forms of XRCC1 in the human population may influence DNA repair and disease susceptibility. The second aim of this research was to demonstrate the applicability of using transgenic cells in a combined study design for determining the biological significance of XRCC1 polymorphisms. Isogenic, mammalian cells lacking XRCC1 were transfected with various forms of the human XRCC1 gene, exposed to different genotoxicants, and assessed for single strand break repair capacity. Only cells expressing the 280His variant showed a repair defect. Subsequently, evaluation of data from the Carolina Breast Cancer Study demonstrated associations between XRCC1 280His, smoking, and breast cancer. Together these studies demonstrate that accessory factors can influence BER efficiency and illustrate the importance of a multi-disciplinary approach for investigating the link between genes, the environment, and disease risk.
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  • In Copyright
Advisor
  • Swenberg, James A.
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Open access
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