Cellular responses to DNA damage and other stresses are important determinants of cell viability and mutagenesis and impact the development of a wide range of human diseases. DNA damage responses, in particular, are major determinants of both cancer development and outcomes of cancer therapies. Induction of signal transduction pathways is a critical aspect of cellular responses to these stresses and significant advances have been made in recent years elucidating the biochemical steps in such signaling pathways. Clarification of such steps enables modulation of these responses, which can enhance research studies and can lead to the generation of new medicines to prevent and treat these diseases. This application describes an enhancement of a novel technique developed in the Kastan laboratory in which DNA double strand breaks (DSBs) are introduced at defined sites in the human genome. Introduction of site-directed DNA damage permits studies of molecular events occurring at and around the DSB and direct assessment of repair (re- ligation) of DNA breaks. The enhancements of this technique permit greater temporal control of introduction of the DSBs and allow assessment of the kinetics and completeness of DSB repair. This enhanced approach will be used in the experiments proposed in this application to elucidate the dynamic changes in protein movements/modifications occurring at and around the DSB and explore factors which may determine efficiency of repair of the DNA breaks. One major focus is exploration of the molecular controls of nucleosome disruption at the DSB site and elucidating the functional importance of this nucleosomal disruption. It is well established that numerous modifications occur in chromatin-associated proteins surrounding DSBs during the damage/repair process;however, the functional role(s) of many of these modifications, especially related to DNA repair, remain to be clarified. Experiments are proposed that will use this novel system to explore the potential impact of these chromatin changes surrounding the DSBs on the ability of the cell to repair (re-ligate) DNA breaks. Further, the mechanisms and impact of the breast cancer susceptibility gene product, Brca1, on this process will be investigated. The proposed studies will provide novel insights into molecular mechanisms associated with the DNA DSB repair process in human cells.
DNA damage and repair are critical determinants of cancer development and responses of tumors to chemotherapy and radiation therapy. Based on a novel system developed to probe molecular events associated with DNA breakage in human cells, studies are proposed to explore factors that affect the ability of human cells to repair DNA double strand breaks. Insights gained into these mechanisms associated with cellular responses to DNA damage will improve our understanding of cancer predisposition and development and have the potential to lead to development of new approaches to enhancing tumor responses to therapy.
|Bakkenist, Christopher J; Kastan, Michael B (2015) Chromatin perturbations during the DNA damage response in higher eukaryotes. DNA Repair (Amst) 36:8-12|
|Goldstein, Michael; Kastan, Michael B (2015) Repair versus Checkpoint Functions of BRCA1 Are Differentially Regulated by Site of Chromatin Binding. Cancer Res 75:2699-707|
|Wang, Qinhong; Goldstein, Michael; Alexander, Peter et al. (2014) Rad17 recruits the MRE11-RAD50-NBS1 complex to regulate the cellular response to DNA double-strand breaks. EMBO J 33:862-77|
|Henriksson, Sofia; Rassoolzadeh, Hanif; Hedström, Elisabeth et al. (2014) The scaffold protein WRAP53? orchestrates the ubiquitin response critical for DNA double-strand break repair. Genes Dev 28:2726-38|
|Goldstein, Michael; Derheimer, Frederick A; Tait-Mulder, Jacqueline et al. (2013) Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair. Proc Natl Acad Sci U S A 110:16874-9|
|Moding, Everett J; Kastan, Michael B; Kirsch, David G (2013) Strategies for optimizing the response of cancer and normal tissues to radiation. Nat Rev Drug Discov 12:526-42|