Direct Analysis of Fork Blockage and DNA Repair in vivo

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DESCRIPTION (provided by applicant): This application focuses on the pathways of replication fork failure and restart, with a particular emphasis on the response of the fork to site-specific lesions in the template. One type of lesion to be analyzed involves inhibitors of DNA topoisomerases. Several important anticancer drugs, as well as the antibacterial quinolones target type II DNA topoisomerases. These inhibitors stabilize the cleavage complex, a reaction intermediate consisting of the enzyme covalently attached at sites of enzyme-mediated DNA breakage. The cleavage complex is necessary but not sufficient for cytotoxicity, and evidence indicates that DNA replication is important for converting cleavage complexes into cytotoxic and potentially mutagenic lesions. We will attempt to decipher the pathway of cytotoxic lesion generation, in both a phage T4 model system for antitumor drug action and in quinolone-treated Escherichia coli. A second type of lesion involves covalent complexes between cytosine methylases and DNA, but in this case, there is no inherent DNA break. Covalent methylase-DNA complexes are induced at the methylase recognition sites by treatment of cells with aza-cytosine-related compounds, which include antitumor agents active against leukemias and other diseases. These compounds have complex mechanisms of action, and the experiments in this application will focus on the consequences of protein-DNA complex formation. We will test the hypothesis that methylase-DNA adducts block the replication fork, and explore the possibility that this fork blockage leads to DNA damage that might be involved in drug cytotoxicity and induced mutagenesis. Finally, abasic sites are a common DNA lesion that occurs under normal growth conditions, and if unrepaired, likely trigger replication fork blockage. We will attempt to deliver DNA with site-specific abasic sites into living E. coli cells, and physically analyze repair and replication fork blockage at these sites.