Understanding cellular responses to DNA damage at the molecular level will provide invaluable insights into questions of both how cancers start and how to cure cancers. The ATM gene product is a major participant in all cellular responses to ionizing irradiation. If ATM function is absent in a cell, it exhibits decreased survival, abnormal cell cycle arrests, and increased chromosomal breakage after irradiation. ATM is a protein kinase and several substrates of this enzyme have recently been identified. A new target of the ATM kinase was identified in the experiments leading up to this application. In response to ionizing irradiation, serine 957 of SMC1 is phosphorylated in cells in an ATM-dependent manner and this same site was found to be the target of ATM kinase in in vitro assays. SMC1 protein has previously been implicated in processes involving chromosomal and DNA dynamics, in particular chromosome cohesion and DNA recombination. Because of its involvement in these processes, it is of particular interest to clarify the functional role(s) of ATM phosphorylation of SMC1 following ionizing irradiation. It is reasonable to predict that loss of ATM phosphorylation of SMC1 on serine 957 may contribute to the radiosensitivity and enhanced chromosomal breakage seen in AT cells. Experiments are proposed that will investigate the functional significance of this phosphorylation event. Numerous cellular responses to ionizing irradiation will be examined, including cell cycle checkpoints, radiosensitivity, chromosomal breakage, and recombination activities. The importance of this site in the biochemical activities of SMC1 will also be examined. Since the lack of an SMC1-null cell line limits some of the functional studies that can be performed, mice in which the SMC1 gene is disrupted by homologous recombination will be generated. Since it is possible that such a mouse will not be viable, a """"""""knock-in"""""""" mouse in which serine 957 of SMC1 will be mutated to alanine (so that it is not able to be phosphorylated by ATM) will also be generated. Significant insights into the role of SMC1 phosphorylation can be gained by studies of the organ development and responses to irradiation as well as from generation of cell lines from these mice.
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