Structural and Biophysical Characterization of the Nucleotide Excision Repair Factor XPA

Abstract

Maintaining the integrity of the genome is critical for the survival of all organisms. Genome maintenance must be efficient because we are constantly under exposure to genotoxic agents including UV-light, endogenous and exogenous reactive oxygen species, and chemical compounds from the environment. These toxins give rise to a variety of DNA lesions ranging from single strand breaks, abasic sites, cross-links and covalent adducts. Persistence of these lesions in the genome can lead directly to apoptosis of the cell or result in mutations, which in turn can lead to carcinogenesis. To combat genetic instability, multiple pathways have evolved to efficiently repair different types of DNA lesions. Nucleotide excision repair (NER) is a repair pathway specialized for removing bulky DNA lesions, typically arising from exposure to sun light, chemical carcinogens in the environment, and certain natural metabolites. Xeroderma pigmentosum complementation group A (XPA) protein is an essential scaffolding protein in the NER machinery. Its importance is underscored by the observation that XPA mutations are frequently associated with severe clinical symptoms of genetic disorder xeroderma pigmentosum (XP). The interaction of XPA with DNA is a core function and a number of mutations in the DNA binding domain are associated with XP disease, suggesting the importance of XPA-DNA interaction in human NER. Although early NMR structures of human XPA and complementary data on DNA binding are available, molecular details of how human XPA binds DNA remain unclear. Moreover, DNA binding of XPA is likely linked to its interaction with another NER factor replication protein A (RPA). This dissertation focused on elucidating the molecular basis of XPA-DNA interaction in the context of human NER. A combination of sequence analysis and a series of DNA binding assays redefined the DNA binding construct of XPA (XPA DBD), which had been misidentified for nearly 20 years. NMR studies identified key residues within the XPA DBD that are involved in interactions with DNA and RPA70AB. Moreover, biochemical studies of mutations within XPA DBD provided initial insights into genotype-phenotype correlations for these mutations and XP disease. Findings from this study enhance the mechanistic understanding of human NER and XP disorders.