Date of Graduation

12-2016

Document Type

Dissertation (PhD)

Program Affiliation

Molecular Carcinogenesis

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Richard D. Wood, PhD

Committee Member

Karen M. Vasquez, PhD

Committee Member

Mark T. Bedford, PhD

Committee Member

Shawn Bratton, PhD

Committee Member

Rick A. Finch, PhD, DABT

Abstract

Nucleotide excision repair (NER) in mammalian cells includes xeroderma pigmentosum group A protein (XPA) as a core factor. XPA and other NER proteins have been detected previously at some active promoters, and NER deficiency is reported to decrease activated transcription of selected genes. To determine the global extent of XPA influence on transcription, we analyzed the human transcriptome by RNA sequencing. We first confirmed that XPA is confined to the cell nucleus even in the absence of external DNA damage, in contrast to previous reports that XPA is normally resident in the cytoplasm and is imported following DNA damage. We then analyzed four genetically matched human cell line pairs deficient or proficient in XPA. At a false discovery rate of 0.05, 325 genes were common in all four pairs with a significant XPA-dependent directional change in gene expression. These genes were highly represented in pathways for the maintenance of mitochondria, metabolism and neurological system. Only 27 genes were regulated by more than 1.5 fold change. The most significant hits were AKR1C1 and AKR1C2, involved in steroid hormone metabolism, and the corresponding proteins were lower in XPA-deficient cells. Transactivation by retinoic acid caused a modest enrichment of genes involved in transcription-related functions in XPA proficient cells. The results show that XPA status significantly influences a small subset of human genes that are important for mitochondrial and metabolic functions. The results may help explain defects in neurological function and sterility in individuals with xeroderma pigmentosum (XP).

An NER deficiency enhances sensitivity of mammalian cells to DNA interstrand crosslinks (ICL)-generating agents. I found that XPA is retained on damaged DNA following exposure to UVA-activated psoralen, and investigated repair of a triplex forming oligonucleotide (TFO)-directed psoralen ICL. A TFO-directed psoralen DNA ICL was constructed in closed-circular DNA. In NER proficient human cell extracts, incisions were detected on both strands of the damaged DNA 3’ to the psoralen ICL. Incision sites on the TFO bound strand were flanked by incision sites 40-42 nucleotides away from the ICL, with incisions 10-12 nucleotides away on the other strand.

Keywords

Nucleotide excision repair, XPA, Interstrand crosslink, Transcription, DNA repair

Available for download on Friday, December 15, 2017

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