Nucleotide excision repair (NER) is the main DNA repair pathway responsible for the removal of bulky DNA lesions induced by UV radiation, environmental mutagens, and chemotherapeutic agents. Deficiencies in NER are associated with the genetic disorder xeroderma pigmentosum (XP), which is characterized by an extreme sensitivity to ultraviolet rays from the sun. XP patients have a 2000-fold increased incidence of skin cancer due to the inability to repair UV-induced DNA lesions. Although the core NER factors and reactions have been discovered, additional studies are required to fully understand the assembly and regulation of NER complexes. Specifically, many relatively weak and dynamic interactions between NER factors and DNA that drive the assembly of the NER complex have yet to be elucidated.<br/><br/>To address this need, we are developing cross-linking mass spectrometry-based techniques (XL-MS) in combination with biochemical and cryo-EM approaches to elucidate all transient DNA-protein and protein-protein interactions formed during the steps of NER assembly. Preliminary XL-MS experiments with purified XPC-RAD23B, which initiates global genome-NER by recognizing and binding to distorted DNA,<sup>1</sup> has identified specific amino acid-amino acid and amino acid-nucleoside interactions between XPC-RAD23B and damaged DNA. After optimization of the XL-MS techniques and analysis, we will investigate the formation of downstream NER complex formation using purified factors. Our studies are expected to provide unprecedented insights towards understanding the mechanisms of dynamic and ordered assembly of NER factors on damaged DNA and the progression through the NER pathway. Furthermore, a more complete understanding of NER assembly is needed for understanding the carcinogenicity of environmental mutagens and could lead to improved anti-tumor treatments.

Studying the assembly of nucleotide excision repair complexes by biochemical and crosslinking mass spectrometry approaches

2019 Fall National Meeting

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