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.