Mechanisms of mismatch repair mediated cell death after alkylating agent exposure

 描述（由申请人提供） 博士伊娃Goellner于2006年获得卡内基梅隆大学化学工程和生物医学工程学士学位。她读的是博士学位。匹兹堡大学医学院罗伯特·索博尔博士实验室的一项研究。她的工作主要集中在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。她的研究生工作导致了癌症研究的第一作者手稿，两个共同第一作者手稿，第一作者评论和几个共同作者的作品，并提供了细胞和分子生物学，哺乳动物细胞培养，药理学和细胞代谢测量的强大背景。Goellner博士于2011年10月加入Richard Kolodner博士的实验室，研究一种单独的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。错配修复需要切除步骤来去除DNA，直到并包括错配，然而，唯一已知的涉及的外切核酸酶Exo 1的缺失不具有所需MMR组分所预期的强增变子表型。这导致存在Exo 1独立和Exo 1依赖的子途径的假设。 Goellner博士获得了NIH NRSA Ruth L。Kirschstein F32博士后奖学金，以筛选特异性破坏Exo 1非依赖性MMR的加载钳PCNA突变。Goellner博士使用酿酒酵母鉴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Exo 1非依赖性MMR的模型。这项工作发表在Molecular Cell的第一作者手稿中，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一个新的模型系统和酵母遗传筛选和生物化学方面的技术专长。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的路德维希癌症研究所进行。路德维希研究所是一个由九个研究实验室组成的多元化研究小组，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。圣地亚哥分支是路德维希研究所中心的全球社区的一部分，其特点是其卓越的研究。这九个实验室共同努力，提供了一个高度协作的环境，几乎任何生物学问题都可以通过内部合作来回答，它提供了一个高质量的研究环境，每个实验室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有着出色的记录，这些博士后研究员在顶级大学获得终身教职， 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UCSD共享的研讨会系列，资源和核心设施，以及与圣地亚哥大量研究机构互动的机会。K99/R 00独立途径提案的重点是回答长期存在的问题，即烷化剂暴露后MMR依赖性细胞死亡的机制。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博士将联合收割机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，利用各种技术和多模型系统来解决这个问题。Goellner博士将鉴定特异性破坏MMR的Exo 1依赖性途径的突变，并将研究它们对修复复制诱导的错配的遗传和生化后果。这也将产生额外的重要突变，用于评价MMR子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R 00独立部分，将使用功能突变分离的集合来研究DNA烷基化损伤后诱导细胞死亡所需的MMR蛋白功能。该提案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，Exo 1独立和Exo 1依赖性MMR子途径发挥什么作用。这项工作将被转化为人类细胞培养，以更好地理解MMR基因突变对人类健康的影响以及它们如何与环境因子相互作用。这将提供有关基因组编辑最新进展的培训，如CRISPR/Cas9技术。这条通往独立的道路不仅将提供关键技术方面的额外培训， 成为一名成功的独立研究人员所需的技能，并实现她的终身职位的目标。这项工作与Kolodner博士或Sobol博士的研究领域有着明显的不同，并将根据Goellner博士的特殊兴趣和培训为她提供一个独特的研究计划。

 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.