Mechanisms of mismatch repair mediated cell death after alkylating agent exposure

 描述（由申请人提供） Eva Goellner 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。匹兹堡大学医学院 Robert Sobol 博士实验室的研究。她的工作主要集中于 DNA 碱基切除修复 (BER) 途径无法完成修复时产生的修复中间体毒性背后的机制。她的工作有助于阐明 BER 修复中间体与细胞代谢的相互作用。她的研究生工作在癌症研究方面发表了一篇第一作者手稿、两篇共同第一作者手稿、一篇第一作者评论和几篇共同作者作品，并在细胞和分子生物学、哺乳动物细胞培养、药理学和细胞代谢测量方面提供了强大的背景。 Goellner 博士于 2011 年 10 月加入 Richard Kolodner 博士的实验室，致力于研究一种独立的 DNA 修复途径，即 DNA 错配修复 (MMR)，该途径可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去除 DNA，直至并包括错配，但是，删除唯一已知的核酸外切酶 Exo1，并不具有所需 MMR 成分所预期的强突变表型。这导致了存在 Exo1 独立子路径和 Exo1 依赖子路径的假设。 Goellner 博士获得了 NIH NRSA Ruth L. Kirschstein F32 博士后奖学金，用于筛选加样夹 PCNA 中的突变，该突变特异性破坏了 Exo1 独立的 MMR。 Goellner 博士利用酿酒酵母确定了许多所需的突变，并通过对它们的研究提供了如何进行不依赖于 Exo1 的 MMR 的模型。这项工作以第一作者手稿发表在《分子细胞》杂志上，第一作者评论和共同作者论文目前正在修订中。她的博士后工作为她提供了酵母遗传筛选和生物化学方面的新模型系统和技术专长。 Goellner 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路德维希癌症研究所进行。路德维希研究所由来自九个研究实验室的多元化研究人员组成，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传导等主题。圣地亚哥分校是路德维希研究所中心全球社区的一部分，其特点是其卓越的研究。这九个实验室共同努力提供一个高度协作的环境，在这个环境中几乎所有的生物学问题都可以通过内部协作来解决，并且它提供了一个高质量的研究环境，每个实验室都定期在最具影响力的期刊上发表文章。路德维希研究所在培养博士后研究员方面拥有出色的记录，这些博士后研究员将在顶级大学获得终身教职，并且在 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 博士可以访问一些共享的 UCSD 研讨会系列、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 独立途径提案的重点是回答烷化剂暴露后 MMR 依赖性细胞死亡机制的长期存在的问题。通过在多个 MMR 基因中使用功能分离突变，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。 Goellner 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，通过多种技术和多个模型系统来解决这个问题。在 K99 奖项的指导部分期间，Goellner 博士将识别特异性破坏 MMR 依赖 Exo1 途径的突变，并将研究它们对修复复制引起的错配的遗传和生化影响。这也将产生额外的重要突变，用于评估对烷化剂敏感性反应的 MMR 子通路。它还将提供生物物理相互作用测定和超分辨率成像等新技术的培训。在 R00 独立部分中，将使用功能突变的分离集合来研究 DNA 烷基化损伤后诱导细胞死亡需要哪些 MMR 蛋白功能。该提案还将测试该反应是否需要下游修复处理和切除，以及如果需要，独立于 Exo1 和依赖于 Exo1 的 MMR 子通路发挥什么作用。这项工作将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。这条获得独立奖的途径将不仅提供关键技术方面的额外培训，而且还提供 成为一名成功的独立研究员并实现终身教授职位目标所需的技能。这项工作明显偏离了 Kolodner 博士或 Sobol 博士的研究领域，并将根据 Goellner 博士的特殊兴趣和培训为她提供独特的研究计划。

 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.