2015 — 2017 |
Freudenthal, Bret D. |
R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
Dna Repair Strategies That Impact Genomic Stability During Oxidative Stress @ University of Kansas Medical Center
DESCRIPTION (provided by applicant) Oxidative stress is induced by environmental exposure to exogenous stressors found in the air we breathe, food we eat, and water we drink. Exposure leads to DNA damage that is linked to pathogenesis of cancer and neurological disorders. The major form of damage is 8-oxo-7,8-dihydro-2'-deoxyguanosine which occurs in both the DNA (8-oxoG) and nucleotide pools (8-oxo-dGTP). The risk posed by 8-oxoG and 8-oxo-dGTP arises from their dual coding potential resulting in non-mutagenic base pairing with cytosine or mutagenic base pairing with adenine during DNA polymerase replication. While DNA polymerases are responsible for mediating the human health impact during oxidative stress, the strategy they use to process oxidative DNA damage remains unclear. To probe these strategies I have developed time-lapse crystallography, permitting an atomic level understanding of how polymerases utilize 8-oxoG. This approach uses natural substrates to capture novel intermediates during the reaction. The candidate hypothesize that processing of oxidative DNA damage by DNA polymerase (pol) Beta alters DNA repair capacity, impacting downstream accessory factors and repair pathway choice. During the K99 phase, under the mentorship of Dr. Samuel Wilson, the candidate will gain essential training in transient-state kinetics while identifying molecular strategies by which pol Beta proofreads opposite 8- oxoG using its reverse reaction (pyrophosphorolysis). This reaction is biologically important to genomic stability and drug resistance. Combining enzymology with time-lapse crystallography will define key intermediates during the proofreading of cytosine or adenine opposite 8-oxoG. This will provide molecular insights to modulate the removal of the mutagenic adenine opposite 8-oxoG to enhance genomic stability or block the removal of chemotherapeutic chain terminating drugs. In the R00 phase, the candidate will determine the molecular mechanisms of DNA polymerase dependent generation and propagation of 8-oxoG. Using a similar approach, he will determine how 8-oxo-dGTP is inserted into DNA and how 8-oxoG is bypassed during replication. This will identify molecular strategies used to process oxidative DNA damage that modulate the mutagenic outcomes during generation and propagation of 8-oxoG. The candidate will further differentiate himself from his mentor by identifying the impact pol Beta strategies have on accessory factors and pathway differentiation during DNA repair. The candidate will determine how pol Beta conformational changes alter substrate channeling to other repair enzymes (e.g., Ape1) and the subsequent processing of 3'-8-oxoG by Ape1. The candidate's comprehensive study on DNA damage processing and the impact on accessory factors will provide a significant advance to our current understanding of the environmental DNA damage response. Additionally, he will gain essential training in transient-state kinetics to complement my structural biology background. These studies fulfill the strategic goals of the NIEHS-NIH by training the next generation of environmental scientists, determining how oxidative DNA damage is processed, the impact it has on larger repair co-complexes, and providing insights into deleterious human health impacts.
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0.976 |
2018 — 2021 |
Freudenthal, Bret D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ape1 Cleavage Mechanisms During Dna Repair @ University of Kansas Medical Center
Principal)Investigator:))Freudenthal,,Bret,D.,, ,, Exposure to environmental hazards induces oxidative stress and promotes deleterious modifications to the structure of DNA. These modifications are potentially mutagenic and can promote numerous human maladies, including cancer. The base excision repair (BER) pathway is the cells primary defense against oxidative DNA damage and maintains genome stability. To this point, genetic polymorphisms and defects in key BER enzymes show up in several human populations, and are often associated with an increased cancer risk. An essential BER enzyme is human apurinic/apyrimidinic (AP) endonuclease 1 (APE1), which is a multifunctional enzyme that processes DNA damage during BER. Utilizing the same active site, APE1 performs both AP endonuclease (endo) and 3? to 5? exonuclease (exo) activities. APE1 endo activity has been rigorously characterized. In contrast, the mechanism for APE1 exo activity remains elusive, and it is unclear how the compact active site can accommodate both an endo substrate (abasic site) and an exo substrate (3? mismatched or damaged base). Moreover, the channeling of toxic DNA intermediates by the BER co-complex during APE1 exo activities remains entirely unstudied, leaving a significant gap in our understanding of BER. Therefore, the objective of this proposal is to determine the APE1 exo mechanism during repair of mismatched and damaged DNA ends. We will place this activity in context of the larger DNA repair co-complex during BER substrate channeling. We hypothesize the exo reaction of APE1 is dependent on unique active site contacts to open the binding pocket during proofreading and the processing of damaged DNA ends. We additionally predict exo substrates promote DNA substrate channeling between APE1 and DNA polymerase beta (the next enzyme in the pathway) during BER. To test this, we propose the following aims: (1) Determine the mechanism of APE1 exo activity during BER proofreading; (2) Determine the mechanism of APE1 catalyzed removal of 3?-PG end damage; and (3) Determine the mechanism of BER substrate channeling during APE1 exo activity. To accomplish these aims we will utilize time-lapse X-ray crystallography to observe catalysis at the atomic level, and pre-steady-state enzyme kinetics to parse out the rates of important steps during catalysis. To address the mechanism of substrate channeling during APE1 exo activity, we will use single-molecule total internal reflection microscopy (TIRFM) to observe the assembly/disassembly of BER complexes on DNA. Small angle neutron scattering will complement the TIRFM studies by determining a structural envelope of the BER co-complex. Using this multidisciplinary approach, we will cast light on previously understudied APE1 DNA repair mechanisms. 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. This approach has proven successful for proteins central to DNA repair pathways, such as PARP-1.
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1 |
2018 — 2021 |
Freudenthal, Bret D. |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Structural and Mechanistic Studies of Dna Repair @ University of Kansas Medical Center
Principal)Investigator:))Freudenthal,,Bret,D.,, ,, 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. In addition, it is poorly understood how deviations in the classical BER pathway affect the DNA repair process and genome stability. These deviancies range from mismatched-, damaged-, and ribo-nucleotides inserted by a DNA polymerase, to the coordinated repair of ?dirty? or damaged DNA ends that block BER. These scenarios become particularly biologically relevant during times where there is an increase in genome instability (i.e., in cancer cells and/or during therapeutic treatments). The overarching goal of this proposal is to understand the molecular mechanisms of each BER component individually and to place these activities within the larger BER co-complex with damaged DNA repair intermediates. Elegant biophysical approaches are required to elucidate these BER complexities and to provide both a foundation for interpreting the biological response and the subsequent 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, neutron crystallography, small angle neutron scattering, molecular dynamic simulations, enzyme kinetics, and single-molecule total internal reflection microscopy. Using these methodologies, we will determine 1) How the location of DNA damage alters the DNA polymerase mechanism during repair; 2) How does the poorly characterized APE1 exonuclease reaction process damaged RNA and DNA repair blocks; 3) What are the mechanistic roles of protons during DNA damage and repair; 4) How are DNA repair complexes formed and structurally organized? 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.
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1 |
2019 — 2020 |
Freudenthal, Bret D. |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Cryoem Structures of Lf-Anthrax Pore Translocon At Ph 5.5 @ University of Kansas Medical Center
Project Summary/Abstract The primary goal of this project is to use cryo-EM single particle analysis to obtain atomic resolution structures (2.2-2.9 Å) of the functional lethal anthrax toxin containing the N terminal domain lethal factor (LFN) bound to the anthrax toxin protective antigen (PA) pore complexes inserted into POPC lipid nanodiscs (LFN-PA pore-nanodiscs). This complex, as well as the PA-pore nanodisc complex, are exposed to translocation competent pH conditions (5.5) and the atomic structures of each of these single LFN or 3 bound LFN to the anthrax pore will be evaluated to uncover the dynamic differences between translocation competent (pH 5.0). These forms will be compared with the inhibited (pH 7.5) forms. Our preliminary structure with a single data collection session on Hong Zhou's Titan Krios yielded a 5.9 A structure that appears to show extra density from the LFN unstructured tail associated with the phe clamp pore lumen. More high resolution data is required to obtain an atomic resolution structures will be obtained from in collaboration with Hong Zhou group (UCLA) (prescreening with Tommi White- MU). We routinely obtain superior solubilized nanodisc (lipid bilayer) complexes in vitreous ice of both 1LFN-PA pore and saturated 3LFN-PA pore complexes to at pH 5.5 enabling us to observe the pH induced changes in atomic structures of these PA pore complexes. Our previously NIH R01 funded structural analysis with an CCD detector (Wah Chiu NCMI site Baylor) culminated in a complete 17 Å 3:1 LFN-PA pore nanodisc complex structure. Acquisition of the desired molecular detail of these initial engagement complexes will specifically provide the atomic snapshots of complexes in various physiologically relevant pH environments. Research and Biotechnology implications: ? Our novel methods to generate cryo-EM ready samples and acquire atomic structure of the PA pore translocon nanodisc complexes should serve as a reasonable template to enhance the acquisition of atomic structure for other aggregation prone membrane proteins inserted into authentic lipid bilayers. ? Our approaches allow us to both control initial ligand association complexes for easier sample preparation of 1:1 and 3:1 (LFN:PA pore) complexes and specific population selection methodologies for cryo-EM analysis. Our research represents the first successes in constructing late endosomal anthrax complexes at pH 5.0 using novel solution protocols. Our approach is highly relevant toward reconstructing other bacterial toxin and viral interactions under late endosome conditions. ? These easy yet powerful approaches of controlled immobilized construction and release are most certainly applicable toward other problem protein constructions where pH dependent changes occur that result in membrane insertion that dictate bacterial toxin and viral entry into the cell.
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0.976 |