Cellular responses to DMAdamage are important determinants of cancer development, outcome following cancer therapies, and the amount of tissue damage occurring after vascular injuries such as myocardial infarction and stroke. In recent years, we have made significant strides in understanding the molecular controls of these damage responses in mammalian cells. While elucidating substrates of the ATM kinase, we made the unexpected observation that SMC1 is phosphorylated by ATM after ionizing radiation (IR). This was unexpected because SMC1 was a known component of the cohesin complex, a protein complex required for sister chromatid cohesion during mitosis, but it was not known to be an important component of signal transduction pathways that facilitate cellular responses to DNA damage. In the previous funding period of this grant we elucidated the functional importance of SMC1 phosphorylation. We demonstrated that it is phosphorylated by ATM on two serines after IR and we implicated these phosphorylation events in modulating the S-phase checkpoint, chromosomal breakage, and radiosensitivity. We also demonstrated that these two serines were phosphorylated by the ATR kinase after other types of DNA damage, thus suggesting a general role for these phosphorylations in cellular stress responses. We generated """"""""knock-in"""""""" mice in which the two phosphorylation sites were mutated and found that cells from these mutant mice exhibited normal activation of ATM and phosphorylation of ATM substrates like NBS1 and BRCA1. Despite these normal signaling steps, the cells exhibited increased chromosomal breakage and an S-phase checkpoint defect. This led to a model in which SMC1 phosphorylation is critical for minimizing chromosomal instability. While exploring the mechanisms by which SMC1 phosphorylation facilitates damage responses, we found two proteins that bound to the phosphorylated form of SMC1: hTRF4 and hEndonuclease V. In this application, we propose experiments to further elucidate the roles of these two proteins in SMC1 binding and DNA damage responses. We also propose experiments using mutant mice and cells that we generated or will generate to further elucidate the exact role(s) that SMC1 and its phosphorylation play in helping cells deal with DNA damage. These studies will help us understand how cells respond to DNA damage and other stresses at the molecular level.
|Derheimer, Frederick A; Kastan, Michael B (2010) Multiple roles of ATM in monitoring and maintaining DNA integrity. FEBS Lett 584:3675-81|
|Rainey, Michael D; Charlton, Maura E; Stanton, Robert V et al. (2008) Transient inhibition of ATM kinase is sufficient to enhance cellular sensitivity to ionizing radiation. Cancer Res 68:7466-74|
|Kastan, Michael B (2008) DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes Memorial Award Lecture. Mol Cancer Res 6:517-24|
|Kastan, Michael B (2007) Our cells get stressed too! Implications for human disease. Blood Cells Mol Dis 39:148-50|
|Bekker-Jensen, Simon; Lukas, Claudia; Kitagawa, Risa et al. (2006) Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 173:195-206|
|Kitagawa, Risa; Bakkenist, Christopher J; McKinnon, Peter J et al. (2004) Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway. Genes Dev 18:1423-38|