Mechanisms of mismatch repair mediated cell death after alkylating agent exposure

烷化剂暴露后错配修复介导的细胞死亡机制

• 批准号: 9761615
• 负责人: Eva Marie Goellner
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761615
• 关键词: ATP phosphohydrolase; ATR gene; Alkylating Agents; Alkylation; Apoptosis; Area; Award; Bachelor' s Degree; Base Excision Repairs; Binding; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Biology; Biomedical Engineering; Biophysics; CRISPR/Cas technology; California; Cell Culture Techniques; Cell Cycle; Cell Cycle Arrest; Cell Death; Cell Line; Cell Survival; Cell model; Cells; Cellular Assay; Cellular Metabolic Process; Cellular biology; Cessation of life; Chemical Engineering; Chromosomes; Closure by clamp; Collaborations; Collection; Communities; Complex; Core Facility; DNA; DNA Alkylation; DNA Damage; DNA Repair Pathway; DNA lesion; Data; Dependence; Development; Doctor of Philosophy; EXO1 gene; Eating; Engineering; Environment; Environmental Tobacco Smoke; Evaluation; Excision; Exonuclease; Exposure to; Fellowship; Food; Futile Cycling; G2/M Arrest; Genetic; Genetic Screening; Genome; Goals; Health; Human; Image; In Vitro; Industrialization; Institutes; Journals; Kinetics; Laboratories; Laboratory Research; Lead; Libraries; MGMT gene; Malignant Neoplasms; Mammalian Cell; Manuscripts; Measurement; Measures; Mediating; Mentors; Metabolism; Methyltransferase; Mismatch Repair; Modeling; Molecular; Molecular Conformation; Molecular and Cellular Biology; Mutation; National Research Service Awards; Nitroso Compounds; Nuclear Extract; Paper; Pathway interactions; Pharmacology; Phase; Phenotype; Phosphotransferases; Plasmids; Play; Positioning Attribute; Postdoctoral Fellow; Process; Proteins; Proteomics; Publishing; Reaction; Reporter; Research; Research Institute; Research Personnel; Resolution; Resources; Role; S-Adenosylmethionine; Saccharomyces cerevisiae; Series; Signal Transduction; Structure; TEL1 Gene; Technical Expertise; Techniques; Technology; Testing; Thymidine; Toxic effect; Training; Translating; United States National Institutes of Health; Universities; Work; Yeasts; anticancer research; base; collaborative environment; endonuclease; environmental agent; exposed human population; gene repair; genome editing; genome integrity; genotoxicity; insight; interest; medical schools; mutant; mutation screening; programs; protein function; public health relevance; reconstitution; recruit; repaired; response; skills; structural biology; success; tenure track; tool; yeast genetics

 DESCRIPTION (provided by applicant) Dr. Eva Goellner received her bachelor's degree in Chemical Engineering and Biomedical Engineering from Carnegie Mellon University in 2006. She did her Ph.D. research at the University of Pittsburgh, School of Medicine in the Laboratory of Dr. Robert Sobol. Her work primarily focused on the mechanisms behind the toxicity of repair intermediates that are generated when the DNA base excision repair (BER) pathway cannot complete repair. Her work helped elucidate the interaction of BER repair intermediates with cellular metabolism. Her graduate work resulted in a first author manuscript in Cancer Research, two co-first author manuscripts, a first author review and several co-author works and provided a strong background in cellular and molecular biology, mammalian cell culture, pharmacology and measurements of cellular metabolism. Dr. Goellner joined the laboratory of Dr. Richard Kolodner in October of 2011 working on a separate DNA repair pathway, DNA mismatch repair (MMR), that repairs mispairs formed during normal replication. Mismatch repair requires an excision step to remove the DNA up to and including the mispair, however deletion of the only known exonuclease involved, Exo1, does not have the strong mutator phenotype expected of a required MMR component. This lead to the hypothesis that Exo1-independent and Exo1-dependent subpathways exist. Dr. Goellner received an NIH NRSA Ruth L. Kirschstein F32 postdoctoral fellowship to perform a screen for mutations in the loading clamp, PCNA, that specifically disrupted Exo1-independent MMR. Using Saccharomyces cerevisiae Dr. Goellner identified a number of the desired mutations and through the study of them provided a model of how Exo1-independent MMR is carried out. This work was published in a first author manuscript in Molecular Cell, a first author review and a co-author paper currently under revision. Her postdoctoral work provided her with a new model system and technical expertise in yeast genetic screens and biochemistry. Dr. Goellner's postdoctoral work and the mentored portion of this award will be carried at the Ludwig Institute for Cancer Research at the University of California School of Medicine Campus. The Ludwig Institute is a diverse group of researchers in nine research laboratories spanning topics of genetics, genome integrity, proteomics, structural biology, chromosome biology, cell biology and cell signaling. The San Diego branch is part of a worldwide community of Ludwig Institute Centers and is characterized by its excellence in research. These nine laboratories work together to provide a highly collaborative environment in which almost any biological problem can be answered through an in-house collaboration, and it provides a high quality research environment, with each laboratory regularly publishing in the highest impact journals. The Ludwig Institute has an excellent track record in producing postdoctoral fellows that go on to tenure track positions at top tier universities, and is strongly devoted to the training and success of its postdoctoral fellows. Additionally, as part of the Ludwig Institute Dr. Goellner has access to a number of shared UCSD seminar series, resources and Core Facilities as well as the opportunity to interact with the large number of research institutes in San Diego. The K99/R00 pathway to independence proposal focuses on answering long-standing questions into the mechanisms of MMR-dependent cell death after alkylating agent exposure. By using separation-of-function mutations in multiple MMR genes she will be able to systematically evaluate this process at a level of mechanistic detail not previously available. Dr. Goellner will combine her unique background in human cell biology, molecular pharmacology, Saccharomyces cerevisiae genetics and biochemistry to approach this question with a wide variety of techniques and multiple model systems. During the K99 mentored portion of the award Dr. Goellner will identify mutations that specifically disrupt the Exo1-dependent pathway of MMR and will investigate their genetic and biochemical consequences on repair of replication induced mispairs. This will also generate additional important mutations to use in the evaluation of MMR sub-pathways in the response to alkylating agent sensitivity. It will also provide training in new techniques such as biophysical interaction assays and super resolution imaging. During the R00 independent portion a collection of separation of function mutations will be used to study which MMR protein functions are required to induce cell death after DNA alkylation damage. This proposal will also test if downstream repair processing and excision are required for this response and if so what roles the Exo1- independent and Exo1-dependent MMR sub-pathways play. This work will be translated into human cell culture in order to better comprehend the human health consequences of mutations in MMR genes and how they interact with environmental agents. This will provide training in recent advances in genome editing, such as CRISPR/Cas9 techniques. This pathway to independence award will provide additional training not only in critical techniques but also in the skills required to be a successful independent researcher and achieve her goal of a tenure track position. This work is a clear departure from Dr. Kolodner or Dr. Sobol's areas of research and will provide Dr. Goellner a unique research program based on her particular interests and training.

 描述(由申请人提供) 伊娃·戈尔纳博士于2006年在卡内基梅隆大学获得化学工程和生物医学工程学士学位。她在匹兹堡大学医学院罗伯特·索博尔博士的实验室完成了博士研究。她的工作主要集中在修复中间体的毒性机制上，修复中间体是当DNA碱基切除修复(BER)途径不能完成修复时产生的。她的工作有助于阐明BER修复中间体与细胞新陈代谢的相互作用。她的毕业工作产生了癌症研究的一篇第一作者手稿、两篇第一作者手稿、一篇第一作者评论和几部合著作品，并提供了细胞和分子生物学、哺乳动物细胞培养、药理学和细胞新陈代谢测量方面的强大背景。戈尔纳博士于2011年10月加入理查德·科洛德纳博士的实验室，研究另一条DNA修复途径--DNA错配修复(MMR)，修复正常复制过程中形成的错配。错配修复需要一个切除步骤来移除直到并包括错配的DNA，然而唯一已知的外切酶Exo1的缺失并不具有所需MMR成分所期望的强烈突变子表型。这导致了一种假设，即存在Exo1依赖和Exo1依赖的亚通路。Goellner博士获得了NIH NRSA Ruth L.Kirschstein F32博士后奖学金，负责筛查加载钳子--增殖细胞核抗原的突变，该突变特别破坏了非依赖于外显子1的MMR。利用酿酒酵母，戈尔纳博士确定了一些所需的突变，并通过对它们的研究，提供了一个外显子1无关的MMR如何进行的模型。这项工作发表在《分子细胞》杂志上的第一作者手稿、第一作者评论和目前正在修改的合著者论文中。她的博士后工作为她提供了一个新的模型系统和酵母基因筛选和生物化学方面的技术专长。戈尔纳博士的博士后工作和该奖项的指导部分将在加州大学医学院校园的路德维希癌症研究所进行。路德维希研究所是一个多元化的研究小组，分布在九个研究实验室，涵盖遗传学、基因组完整性、蛋白质组学、结构生物学、染色体生物学、细胞生物学和细胞信号等主题。圣地亚哥分部是路德维希研究所中心全球社区的一部分，其特点是在研究方面表现出色。这九个实验室通力合作，提供了一个高度协作的环境，在这个环境中，几乎任何生物问题都可以通过内部协作来回答，它还提供了一个高质量的研究环境，每个实验室定期在影响最大的期刊上发表文章。路德维希研究所在培养继续在一流大学担任终身教职的博士后研究员方面有着出色的记录，并具有强大的 致力于博士后研究员的培训和成功。此外，作为路德维希研究所的一部分，Goellner博士可以访问加州大学圣迭戈分校的一系列共享研讨会、资源和核心设施，并有机会与圣地亚哥的大量研究机构互动。K99/R00途径独立的建议集中于回答长期存在的问题，即烷化剂暴露后MMR依赖的细胞死亡机制。通过使用多个MMR基因中的功能分离突变，她将能够在以前无法获得的机制细节水平上系统地评估这一过程。戈尔纳博士将结合她在人类细胞生物学、分子药理学、酿酒酵母遗传学和生物化学方面的独特背景，用各种技术和多种模型系统来解决这个问题。在K99奖项的指导部分，Goellner博士将识别专门破坏MMR依赖于Exo1途径的突变，并将研究它们在修复复制诱导的错配方面的遗传和生化后果。这也将产生额外的重要突变，用于评估MMR亚途径对烷化剂敏感性的反应。它还将提供生物物理相互作用分析和超分辨率成像等新技术方面的培训。在R00独立的部分，功能突变的分离将被用来研究在DNA烷基化损伤后，哪些MMR蛋白功能是诱导细胞死亡所必需的。这项建议还将测试这种反应是否需要下游修复、加工和切除，如果需要的话，外显子通路和外显子通路依赖的MMR亚通路发挥什么作用。这项工作将被转化为人类细胞培养，以便更好地理解MMR基因突变对人类健康的影响，以及它们如何与环境因素相互作用。这将提供有关基因组编辑最新进展的培训，例如CRISPR/CAS9技术。这个独立之路奖不仅将在关键技术方面提供额外的培训，而且还将在 成为一名成功的独立研究人员并实现终身教职的目标所需的技能。这项工作明显偏离了科洛德纳博士或索博尔博士的研究领域，并将根据戈尔纳博士的特殊兴趣和培训为她提供独特的研究计划。