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Project 2: Replication Fork Repair and Signaling

项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

未公开

项目受资助机构

UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB

立项年度

2018

立项时间

未公开

项目编号

5P01CA092584-18

研究期限

未知 / 未知

项目级别

国家级

受资助金额

未知

学科

Cancer Genetics

学科代码

未公开

基金类别

Non-SBIR/STTR RPGs

关键词

未公开

参与者

CORTEZ, DAVID K

参与机构

NATIONAL CANCER INSTITUTE

项目标书摘要：Project 2 ? Replication Fork Repair PROJECT SUMMARY/ABSTRACT Despite active repair and proofreading mechanisms, the replication machinery encounters unrepaired lesions and other forms of replication stress every cell division cycle. Therefore, completing DNA replication faithfully requires specialized replication stress response and error correction mechanisms. Replication fork remodeling by DNA translocases and nucleases can stabilize and repair damaged replication forks while mismatch repair enzymes can correct polymerase errors. However, inactivation or improper regulation of these enzymes generates DNA sequence changes that fuel cancer development. For example, inactivation of mismatch repair (MMR) is the most frequent cause of inherited cancers. Furthermore, oncogenes generate elevated levels of replication stress. While the genetic instability that results from these changes can promote tumorigenesis, it also makes cancer cells more dependent on the remaining replication stress response and repair pathways. Thus, these properties of cancer cells provide therapeutic opportunities that can be exploited by both traditional chemotherapeutic and radiation therapies that target DNA and newer agents like PARP inhibitors that more selectively utilize synthetic lethality to kill cancer cells. The guiding principle of this project is that understanding how replication stress and fork repair activities work in normal and cancer cells is critical to understand both the etiology of cancer and to develop and deploy new therapies. A five-member team of investigators with expertise spanning structural biology, biochemistry, biophysics, genetics, and cell biology will focus on the key regulatory nodes that direct replication-associated repair activities. We will capitalize on the progress made in the last funding period, the ongoing research in project member laboratories, and the synergy created by employing multiple experimental approaches to address the following specific aims: (1) define the mechanisms by which the fork remodeling proteins ZRANB3, HLTF, and SMARCAL1 repair damaged replication forks; (2) define the unique replication-associated cellular functions of fork remodeling proteins; and (3) define mechanisms controlling nuclease activities at replication forks. Collaborations with other SBDR projects will ensure the highest impact of our studies. For example, we will work with project 1 to understand RPA function, project 3 to examine activities of HRR proteins in fork repair, project 4 to explore the mechanism of action of PARP inhibitors, and project 5 to examine the role of MRN proteins at replication forks.

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  • 1.Robust Production,Crystallization,Structure Determination,and Analysis of[Fe-S]Proteins:Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes

    • 关键词：
    • IRON-SULFUR CLUSTER; COLI NITRATE REDUCTASE; X-RAY-SCATTERING; ESCHERICHIA-COLI; DNA-REPAIR; CRYSTAL-STRUCTURE; ENDONUCLEASE-III; ETHYLBENZENE DEHYDROGENASE; ASSEMBLY COMPLEX; GENE-CLUSTER
    • Tsai, Chi-Lin;Tainer, John A.
    • 《FE-S CLUSTER ENZYMES, PT B》
    • 2018年
    • 会议

    [Fe-S] clusters are essential cofactors in all domains of life. They play many biological roles due to their unique abilities for electron transfer and conformational control. Yet, producing and analyzing Fe-S proteins can be difficult and even misleading if not done anaerobically. Due to unique redox properties of [Fe-S] clusters and their oxygen sensitivity, they pose multiple challenges and can lose enzymatic activity or cause their component proteins to be structurally disordered due to [Fe-S] cluster oxidation and loss in air. Here we highlight tested protocols and strategies enabling efficient and stable [Fe-S] protein production, purification, crystallization, X-ray diffraction data collection, and structure determination. From multiple high-resolution anaerobic crystal structures, we furthermore analyze exemplary data defining [Fe-S] clusters, substrate entry, and product exit for the functional oxidation states of type II molybdo-bis(-molybdopterin guanine dinucleotide) ( Mo-bisMGD) enzymes. Notably, these enzymes perform electron shuttling between quinone pools and specific substrates to catalyze respiratory metabolism. The identified structure-activity relationships for this enzyme class have broad implications germane to perchlorate environments on Earth and Mars extending to an alternative mechanism underlying metabolic origins for the evolution of the oxygen atmosphere. Integrated structural analyses of type II Mo-bisMGD enzymes unveil novel distinctive shared molecular mechanisms for dynamic control of substrate entry and product release gated by hydrophobic residues. Collective findings support a prototypic model for type II Mo-bisMGD enzymes including insights for a fundamental molecular mechanistic understanding of selectivity and regulation by a conformationally gated channel with general implications for [Fe-S] cluster respiratory enzymes.

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