Nucleotide excision repair (NER) removes damage to DNA that arises from exposure to environmental agents and solar UV radiation. Defects in NER lead to the inherited disorder xeroderma pigmetosum, which results in a high incidence of skin cancer and in severe cases neurological and developmental defects. NER is also is also critical for the development of resistance to antitumor agents such as cisplatin that function by damaging DNA. NER is therefore a potential target for developing more effective anticancer therapeutic strategies. In this project, we will capitalize on the strengths of our team in the areas of chemistry, biophysics, biochemistry, structural and cell biology to characterize the progressive assembly of NER proteins to recognize, verify and process the damaged DNA. Progress during SBDR3 provided the impetus to conceive this new project. New protein expression strategies allow for the generation of milligram quantities of large protein complexes, in particular TFIIH, required for structural studies. Furthermore, the development of new SAXS approaches and implementation of state of the art cryo-EM methods provides us with the tools for structural characterization of NER complexes. We are now in a position to elucidate in detail how key protein- protein interactions drive the NER reaction, and, using assays to probe for multiple DNA repair pathways, to assess how disruption of such interactions might be exploited to specifically target NER. We will characterize the dynamic mechanisms of NER incision complex assembly with a focus on the interactions among the proteins involved that drive them.
Aim 1 will probe how interactions between XPC- RAD23B and TFIIH mediate damage recognition and verification and elucidate the structural rearrangements that lead to the engagement of TFIIH with the lesion.
In Aim 2 we will investigate how essential dynamic interactions of the XPA scaffold protein with DNA, RPA, TFIIH and ERCC1 guide assembly of the NER pre- incision complex.
In Aim 3, we will generate and structurally characterize the full NER incision complex and define how the recruitment of the two endonucleases ERCC1-XPF and XPG remodels the topology of the damaged DNA substrate to trigger the incision reactions. We anticipate that our studies will provide unprecedented insights into the inner workings of the NER machine and define protein-protein interactions that may serve as viable targets to develop inhibitors to suppress NER during anticancer therapy. Our studies make extensive use of the EMB and SCB cores and are highly integrated with the other SBDR projects. In particular, we will explore how mutations in NER proteins affect other DNA repair pathways and work collaboratively with Projects 2-5 to advance global understanding of the cellular response to DNA damage on specific questions such as the differences in coordination of RPA and XPG activities in damage repair by NER and homologous recombination pathways.
Project 1 ? Nucleotide Excision Repair PROJECT NARRATIVE The understanding of the human nucleotide excision repair (NER) pathway is of great importance for cancer etiology and cancer therapy. Patients with mutations in NER genes suffer from the inherited skin cancer prone disorder xeroderma pigmentosum, yet NER also contributes to the resistance to treatment with numerous agents used in cancer therapy that act by damaging DNA. Through structural and functional studies, we seek to define the complex molecular mechanisms of NER so that they may be targeted to develop improved approaches to antitumor therapy.
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