Abstract
Nucleotide excision repair (NER) is the main DNA repair pathway dealing with bulky adducts formed by UV irradiation and environmental carcinogens. The importance of NER is underscored by the inherited disorder xeroderma pigmentosum (XP) caused by defects in NER genes and characterized by an enhanced predisposition to skin cancer. Of over 30 proteins involved in NER, XPC-RAD23B is responsible for the recognition of diverse substrates, binding the non-damage strand of DNA and probing for the thermodynamic destabilization induced by the lesion. The helicase subunits in TFIIH are likely candidates to scan DNA to confirm the existence of a damage on DNA. Subsequent to the recognition and verification steps, a 24-32nt size oligonucleotide containing the lesion is excised. It has been found that the higher degrees of duplex destabilization induced by a lesion result in a higher repair rate. However, there are still many unresolved questions in understanding the correlation between the structure of a DNA lesion and its processing by NER. Research in this thesis focused on various aspects of damage recognition in human NER, aming to understand how the structure of a lesion affects NER, acetylamino fluorene (AAF) and aminofluorene (AF) adducts of DNA, which create distinct degrees of distortion in various sequence contexts in duplex DNA were synthesized. These substrates were incorporated into plasmids and used in NER assay to study the repair efficiency in cell extracts, in binding assays to measure affinity to XPC-RAD23B, and in thermodynamic studies to measure the extent of DNA destabilization. These experiments showed that the degrees of destabilization of DNA duplex induced by AAF and AF indeed correlated with XPC-RAD23B binding affinity and NER efficiency. Similar experimental methods were applied to DNA lesions induced by another mutagen, aristolochic acid (AA). A previous cellular study had suggested that AA is repaired by transcription coupled (TC) NER, but not global genome (GG) NER. Our studies showed that dA-ALII adducts of DNA are not repaired by GG-NER and not bound specifically by XPC-RA23B, providing a rationale for why ALII adducts persist in human cells. Traditional NER assays require the use of radioactive materials to detect products and have the limitation that only products but not intermediates and unreacted substrates can be detected. To overcome this limitation, click chemistry was used to prepare fluorescently labeled NER substrates and these yielded promising first results in monitoring NER reactions. These fluorescent substrates should facilitate NER studies, by reducing assay time, to detect various NER reaction intermediates and to study NER reactions in a quantitative fashion.
