Investigations Into Dynamic DNA Recognition and Processing During Eukaryotic Nucleotide Excision Repair

DNA undergoes continuous damage from endogenous and environmental sources. Unrepaired DNA damage interferes with cellular functions such as replication and transcription, often resulting in mutations and genome instability. Nucleotide excision repair (NER) is a major DNA repair pathway that removes structurally diverse lesions caused by UV, fuel combustion, industrial pollutants, cigarette smoke, etc. in DNA.

Defects in NER can cause hyper-UV sensitivity and increase mutations that lead to diseases including cancers in humans. This project aims to obtain detailed molecular and structural understanding of critical steps in the initiation of NER. Technical innovations in this project will be applicable to many other systems involving complex and dynamic protein-DNA interactions.

The research will provide cross-disciplinary training opportunities for undergraduate and graduate researchers and enhance the biomedical research environment at Baylor University through close collaboration with the University of Pennsylvania medical school. Outreach activities will include participation in an Advanced Instrumentation Workshop at Baylor, held for faculty and students from local colleges/universities with limited access to high-level instrumentation; development of a research-based biology curriculum ‘Guardians of the Genome’ in collaboration with a high school in Texas, and public demonstrations through the NSF-funded ‘Portal to the Public’ program at Baylor’s Mayborn Museum.

NER is an essential genome maintenance mechanism whose molecular machinery is conserved in all eukaryotes ranging from yeast to humans. Though much is known about the biochemical steps of NER, little is known at the structural level due to challenges in preparing key multiprotein complexes (e.g., TFIIH and Rad4 (yeast homolog of mammalian XPC)) on suitable NER lesions for structural studies.

Building on the team’s prior successes and by combining cryo-electron microscopy (cryo-EM), cross-linking/mass spectrometry (XL-MS), and fluorescence lifetime (FLT)-based conformational analyses as well as yeast genetics, this project aims at comprehensive understanding of NER initiation through dynamic 3-D views of the complex structures and their transitions. Specifically, the project will address: (1) How TFIIH-Rad4 first starts unwinding the DNA on bona fide NER lesions, (2) How holoTFIIH is activated for NER by disengaging the transcription-specific TFIIK kinase module, and (3) How the TFIIH-Rad4 complex transitions to lesion verification stage.

This research using the yeast system will also open new doors to understanding various NER-linked phenotypes in humans and shed light on how NER may be modulated in vivo.

This project is jointly funded by the Genetic Mechanisms and Molecular Biophysics programs of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.