Ionizing radiation results in the cessation of the initiation of DNA replication. DNA single and double stranded breaks and DNA-protein crosslinks are major classes of damage which may contribute to the block of DNA replication. We propose to study the covalent DNA protein crosslinks (DPC) which occur after ionizing radiation and discover whether they represent nuclear structural proteins that could constitute a mechanism whereby DNA replication is halted. The research plan includes the biochemical characterization of the protein and DNA involved in the DPC. The proteins will be analyzed with respect to molecular weight, isoelectric point, and peptide mapping. The purported nuclear matrix proteins and chromosomal attachment proteins have been described and will be compared to the DPC proteins. The DNA will be tested for its size and heterochromicity. A long-range goal of this study is to produce antibody directed against the purified protein(s) involved in the DPC. The antibody or antibodies are important for determining the localization of the DPC within an intact cell, providing the means to generate a specific probe for the DPC and contributing to the purification of the proteins and DNA in the DPC under non-denaturing conditions. The biological features associated with the DPC production include investigating the metabolic turnover of the protein(s), and the dependence of the DPC upon protein and RNA synthesis. Further, AT cells, which do not halt DNA synthesis after ionizing radiation will be used to test the suggested link between the covalent DPC production and the initiation of DNA synthesis. These proposed studies will contribute to the knowledge of DNA-protein crosslinks and may provide a plausible mechanism whereby DNA replication is halted by ionizing radiation.
