Mechanisms of mismatch repair mediated cell death after alkylating agent exposure

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 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.