Structural and Mechanistic Studies of DNA Repair

DNA修复的结构和机制研究

基本信息

  • 批准号:
    10622967
  • 负责人:
  • 金额:
    $ 46.5万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2018
  • 资助国家:
    美国
  • 起止时间:
    2018-09-01 至 2028-08-31
  • 项目状态:
    未结题

项目摘要

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.
氧化应激是一种普遍的和危险的细胞状况,导致对细胞的有害修饰。 DNA的结构。这些修饰促进了诱变,并因此促进了许多突变体的发展。 包括癌症在内的人类疾病。碱基切除修复(BER)途径是细胞对DNA的主要防御途径。 氧化DNA损伤,是基因组稳定性的重要监护人。虽然单个酶的作用, 经典的BER循环基本上已经建立,但这些酶如何在一个特定的环境中一起发挥作用仍然是个谜。 多蛋白质/DNA复合物,以促进每个蛋白质之间的毒性DNA修复中间体的通道。 重要的是,BER不仅发生在裸双链DNA上,而且发生在由 核小体这些核小体为BER酶进入和有效修复DNA提供了屏障 损害DNA修复蛋白克服这一障碍修复DNA损伤的机制, 对核小体了解甚少。这项建议的主要目标是了解分子机制 使用裸双链体DNA单独地和在较大的多蛋白质/DNA复合物内的每个BER因子 和染色质;并破译端粒酶复制端粒的分子机制。优雅 生物物理方法需要阐明这些BER的复杂性,并提供基础, 解释生物反应和治疗方法的发展。我们处于得天独厚的有利地位 基于我在DNA损伤和修复方面的良好记录,为了推进这一科学前沿, 合作者和多学科的方法。为了实现这一目标,我们利用时间的综合方法- X射线晶体学,分子动力学模拟,酶动力学,单分子全内 反射显微镜和冷冻电镜使用这些方法,我们将确定1)如何进行新的基本面 机械步骤改变DNA聚合酶和端粒酶机制; 2)如何个别BER酶 组装成一个多蛋白质/DNA复合物,以促进有毒DNA中间体的通道; 3)如何 多蛋白质/DNA BER复合物的结构组织; 4)DNA损伤是如何识别和修复的 核小体;以及5)多蛋白质/DNA BER复合物如何在含有DNA的核小体上形成 损害这组问题将从原子级的关键BER组件的机械理解 在整个BER多蛋白复合物中的结构和动态相互作用。通过这样做,我们将奠定 该基金会旨在解决建立细胞模型和开发新的治疗方法的固有挑战, 针对DNA修复的治疗。有了这些信息,我们将更接近我们的长期目标, 为合理的药物设计提供基础,以开发更有效的化疗药物, 靶向参与DNA损伤反应的蛋白质的协同药物组合。

项目成果

期刊论文数量(11)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Mechanism of Deoxyguanosine Diphosphate Insertion by Human DNA Polymerase β.
  • DOI:
    10.1021/acs.biochem.0c00847
  • 发表时间:
    2021-02-09
  • 期刊:
  • 影响因子:
    2.9
  • 作者:
    Varela FA;Freudenthal BD
  • 通讯作者:
    Freudenthal BD
Specific mutations in the D1-D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors.
  • DOI:
    10.1038/cddiscovery.2017.65
  • 发表时间:
    2017
  • 期刊:
  • 影响因子:
    7
  • 作者:
    Bastola P;Wang F;Schaich MA;Gan T;Freudenthal BD;Chou TF;Chien J
  • 通讯作者:
    Chien J
Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase β during base excision repair.
  • DOI:
    10.1016/j.jbc.2023.104636
  • 发表时间:
    2023-05
  • 期刊:
  • 影响因子:
    4.8
  • 作者:
    Fairlamb, Max S.;Spies, Maria;Washington, M. Todd;Freudenthal, Bret D.
  • 通讯作者:
    Freudenthal, Bret D.
History of DNA polymerase β X-ray crystallography.
  • DOI:
    10.1016/j.dnarep.2020.102928
  • 发表时间:
    2020-09
  • 期刊:
  • 影响因子:
    3.8
  • 作者:
    Whitaker AM;Freudenthal BD
  • 通讯作者:
    Freudenthal BD
Anthrax toxin translocation complex reveals insight into the lethal factor unfolding and refolding mechanism.
  • DOI:
    10.1038/s41598-021-91596-3
  • 发表时间:
    2021-06-22
  • 期刊:
  • 影响因子:
    4.6
  • 作者:
    Machen AJ;Fisher MT;Freudenthal BD
  • 通讯作者:
    Freudenthal BD
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Bret D Freudenthal其他文献

Bret D Freudenthal的其他文献

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{{ truncateString('Bret D Freudenthal', 18)}}的其他基金

APE1 Cleavage Mechanisms during DNA Repair
DNA 修复过程中 APE1 切割机制
  • 批准号:
    10443576
  • 财政年份:
    2018
  • 资助金额:
    $ 46.5万
  • 项目类别:
Structural and Mechanistic Studies of DNA Repair
DNA修复的结构和机制研究
  • 批准号:
    9762147
  • 财政年份:
    2018
  • 资助金额:
    $ 46.5万
  • 项目类别:
APE1 Cleavage Mechanisms during DNA Repair
DNA 修复过程中 APE1 切割机制
  • 批准号:
    10202601
  • 财政年份:
    2018
  • 资助金额:
    $ 46.5万
  • 项目类别:
Structural and Mechanistic Studies of DNA Repair
DNA修复的结构和机制研究
  • 批准号:
    10247705
  • 财政年份:
    2018
  • 资助金额:
    $ 46.5万
  • 项目类别:
DNA Repair Strategies that Impact Genomic Stability During Oxidative Stress
氧化应激期间影响基因组稳定性的 DNA 修复策略
  • 批准号:
    9330157
  • 财政年份:
    2015
  • 资助金额:
    $ 46.5万
  • 项目类别:
DNA Repair Strategies that Impact Genomic Stability During Oxidative Stress
氧化应激期间影响基因组稳定性的 DNA 修复策略
  • 批准号:
    9131846
  • 财政年份:
    2015
  • 资助金额:
    $ 46.5万
  • 项目类别:
DNA Repair Strategies that Impact Genomic Stability During Oxidative Stress
氧化应激期间影响基因组稳定性的 DNA 修复策略
  • 批准号:
    9136220
  • 财政年份:
    2015
  • 资助金额:
    $ 46.5万
  • 项目类别:
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