XPC Haplotypes Alter DNA Repair Capacity and Levels of Genetic Damage
Rondelli, Catherine Michelle
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Xeroderma pigmentosum complementation group C (XPC) is the key recognition factor of DNA damage in global genome nucleotide excision repair (NER). The disease Xeroderma pigmentosum (XP) results from mutations leading to structural defects of the encoding gene and in some instances trace to changes in a single nucleotide. The XPC gene is highly polymorphic and while polymorphisms in general have no discernible phenotypic effects, some can alter the structure and function of the encoded protein. To date, the effect of single nucleotide polymorphisms (SNPs) in XPC have not been properly characterized. Documented associations exist between only a few XPC SNPs and cancer risk, leaving a majority of SNPs untested. My hypothesis is that specific XPC SNP combinations (haplotypes) alter DNA repair capacity and levels of genetic damage by altering transcriptional processes and/or protein function. I used bioinformatics to conduct a comprehensive haplotype analysis of the entire genomic sequence of XPC and characterize the effect of haplotypes on genetic damage in a population of smokers as an environmentally exposed population. All genomic region XPC polymorphisms with a minor allele frequency (MAF) ≥0.05, from the HapMap CEPH population were analyzed using PHASE, generating a series of likely phylogenetically clustered haplotypes. Cigarette smokers and matched non-smokers from a White, non-Hispanic population residing in the Houston-Galveston area were genotyped and recoded for these haplotype groups. Association between smoking status and DNA damage was determined using chromosomal aberrations as a biomarker. To characterize the biological effects of the XPC haplotypes, I determined how they affected DNA damage and repair capacity over time (i.e. the genotype/phenotype relationship) using representative cell lines. I evaluated the effect of these haplotypes on NER capacity using ELISA (Enzyme Linked Immunosorbent Assay) following exposure to ultraviolet (UV) radiation. I characterized the functional significance of XPC haplotypes by determining the effects of these haplotypes on transcriptional processing and stability using real-time analysis, and protein expression and stability with Western blot analysis. I found that the haplotypes not only conferred differential repair capacity, but that they did so through uniquely different mechanisms.