Ionizing radiation like a variety of other damaging agents introduces a spectrum of lesions into DNA. It is now evident that mammalian cells respond to such radiation damage by the induction of specific genes, and by the synthesis of a range of proteins to protect the cell or to initiate the pathways of apoptosis or programmed cell death in some cell types. In addition to de novo protein synthesis, activation of pre- existing proteins has also been described in response to DNA damage. The overall aim of this study is to identify proteins induced/activated by ionizing radiation, to clone the genes specifying these proteins, to study their regulation and to relate the functions of these proteins to cell cycle arrest. Parallel studies will be carried out with cells from patients with the human genetic disorders ataxia-telangiectasia, a syndrome characterized by radiosensitivity and predisposition to cancer. The grant will concentrate on: 1(a). Isolation and purification of DNA-binding and associated proteins induced/activated by ionizing radiation. Evidence has been presented recently for the activation of a DNA binding protein by ionizing radiation. This proposal is designed to identify and purify other DNA- binding proteins induced/activated by ionizing radiation using a random set of oligodeoxynucleotides. Protein activation will be compared using gamma-radiation and high energy neutrons. (b) Cloning and characterization of genes specifying DNA binding proteins: Three approaches will be used to isolate cDNAs corresponding to the binding proteins (i) protein sequence data of synthesize PCR primers for amplification of mRNA (ii) Use of specific binding sequence to isolate clones from an expression library (iii) Production of antibodies against proteins and subsequent screening of a cDNA expression library. (c). Identification of target genes 2 . Identification and cloning of genes induced by ionizing radiation This approach will be used as a complementary method to that described in Aim 1 where pre-existing proteins are being studied. Newly or differentially expressed genes will be identified by differential display and further characterized. 3. To isolate genes and identify proteins modifications associated with radiation-induced cell cycle arrest: Recent results demonstrate that ataxia-telangiectasia cells fails to show initial delay transitions between G1/S and G2/M phases and in progression through these phases, but in the longer term after passage through S phase experience a prolonged delay in G2/M. Differential display will also be used to identify genes involved in these cell cycle transitions post-irradiation. Modification of proteins know to be involved in cell cycle passage will also be determined.
