# Structural and Mechanistic Studies of DNA Repair

> **NIH NIH R35** · UNIVERSITY OF KANSAS MEDICAL CENTER · 2024 · $465,000

## Abstract

Oxidative stress is a prevalent and dangerous cellular condition resulting in deleterious modifications to the
structure of DNA. These modifications promote mutagenesis and consequently the development of numerous
human maladies, including cancer. The base excision repair (BER) pathway is the cells primary defense against
oxidative DNA damage and is a vital guardian of genome stability. While the roles of individual enzymes during
a classical BER cycle are largely established, it remains enigmatic how these enzymes function together in a
multi-protein/DNA complex to facilitate the channeling of toxic DNA repair intermediates between each protein.
Importantly, BER not only occurs on naked duplex DNA, but also within chromatin that is composed of
nucleosomes. These nucleosomes present a barrier to BER enzymes accessing and effectively repairing DNA
damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the
nucleosome is poorly understood. The major goals of this proposal are to understand the molecular mechanisms
of each BER factor both individually and within larger multi-protein/DNA complexes using naked duplex DNA
and chromatin; and to decipher the molecular mechanism by which telomerase replicates the telomere. Elegant
biophysical approaches are required to elucidate these BER complexities and to provide both a foundation for
interpreting the biological response and the development of therapeutic treatments. We are in a unique position
to advance this scientific front based on my strong track record in DNA damage and repair, assembled team of
collaborators, and multidisciplinary approach. To meet this goal, we utilize a comprehensive approach of time-
lapse X-ray crystallography, molecular dynamic simulations, enzyme kinetics, single-molecule total internal
reflection microscopy, and cryo-EM. Using these methodologies, we will determine 1) how do new fundamental
mechanistic steps alter the DNA polymerase and telomerase mechanism; 2) how do individual BER enzymes
assemble into a multi-protein/DNA complex to facilitate the channeling of toxic DNA intermediates; 3) how are
multi-protein/DNA BER complexes structurally organized; 4) how is DNA damage identified and repaired within
nucleosomes; and 5) how are multi-protein/DNA BER complexes formed on nucleosomes containing DNA
damage. This set of questions will go from an atomic level mechanistic understanding of key BER components
to the structural and dynamic interactions within the entire BER multi-protein complex. By doing this, we will lay
the foundation to address an inherent challenge in establishing cellular models and developing new therapeutic
treatments that target DNA repair. With this information in hand, we will be closer to our long-term goal of
providing a basis for rational drug design towards the development of more effective chemotherapeutics and
synergistic drug combinations that target proteins involved in the DNA damage response.

## Key facts

- **NIH application ID:** 10919758
- **Project number:** 5R35GM128562-07
- **Recipient organization:** UNIVERSITY OF KANSAS MEDICAL CENTER
- **Principal Investigator:** Bret D Freudenthal
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $465,000
- **Award type:** 5
- **Project period:** 2018-09-01 → 2028-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10919758

## Citation

> US National Institutes of Health, RePORTER application 10919758, Structural and Mechanistic Studies of DNA Repair (5R35GM128562-07). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10919758. Licensed CC0.

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