Integrative Modeling of Biomolecular Machinery in Nucleotide Excision Repair - PROJECT SUMMARY/ABSTRACT Nucleotide excision repair (NER) is an essential genome maintenance pathway that detects and removes harmful DNA lesions resulting from exposure to environmental carcinogens, toxins, alkylating agents, reactive oxygen species and ultraviolet radiation. NER stands out among all DNA repair pathways for its ability to remove the widest array of structurally unrelated lesions. The need to process a wide variety of damaged sites has given rise to a remarkably complex molecular machinery. Defects in this machinery provide a paradigm for the diverse clinical consequences of DNA damage and are associated with severe human diseases – 1) ultraviolet radiation- sensitive syndrome; 2) xeroderma pigmentosum, characterized with extreme cancer predisposition; 3) cerebro- oculo-facio-skeletal syndrome; 4) trichothiodystrophy; and 5) Cockayne syndrome, associated with premature ageing and accelerated neurodegeneration. Furthermore, NER is intricately intertwined with other vital pathways that orchestrate the expression and repair of genes. Thus, understanding the molecular mechanisms of NER is a grand challenge in biomedical science. Progress toward this goal has been hindered by the size, complexity and dynamic nature of the assemblies that accomplish NER. To overcome this critical barrier to progress, we will employ integrative modeling methods, combining state-of-the-art computation with experimental data from cryo-electron microscopy (cryo-EM), site-directed mutagenesis, crosslinking mass spectrometry (XL-MS), hydrogen deuterium exchange (HDX) mass spectrometry and small angle X-ray scattering (SAXS) to elucidate the assembly, function and regulation of key NER complexes. Specifically, our focus is on transcription factor IIH (TFIIH) as the centerpiece of the NER machinery. In Aim1, we will elucidate the functional dynamics of TFIIH and discover key allosteric residue networks enabling the function of this recognized NER master coordinator. We will also decipher the effects of TFIIH disease mutations, providing a novel paradigm for the diverse clinical manifestations of NER impairment. In Aim2, we will unravel the mechanisms of TFIIH-associated lesion scanning and DNA damage verification. In Aim3, we will synthesize diverse structural data to create an integrative model of the most crucial intermediate in NER – the pre-incision complex. Hybrid models will define the structural elements allowing TFIIH to serve as a mobile platform for the assembly and remodeling of the NER machinery. Our work will benefit from synergistic collaborative interactions with world-class experimental groups to inform, validate, and extend our models. Parallel computational and experimental advances will yield key insights into the structure, dynamics and function of NER complexes while making direct connection to genetic disease phenotypes. Success of the project will thus have major impacts - both in understanding disease etiology and in offering a structural framework to devise effective treatments.
