This application seeks continued support for mechanistic studies on the elucidation of the chemistry and biology of DNA cleavage by radical- generating drugs. Efforts in the past few years funded by this proposal have focused on designing new general methods to investigate the mechanism of these DNA cleavers and have been highly successful in unraveling the mechanism of the prototypical DNA cleaver bleomycin (BLM). The BLMs are a class of antitumor antibiotics isolated from S. verticillus that are used clinically against head and neck cancer and testicular cancer. Thus, a thorough understanding of its mechanism of action is of considerable relevance to the development of new chemotherapeutic agents. Work during the past funding period has laid the foundation for the comprehensive studies planned. In the next funding period, the following specific aims will be pursued: 1) The parameters responsible for the sequence, base and conformational specificity of BLM-mediated DNA cleavage will be examined. Methods employed will include isotope effect analysis and alternate nucleic acid structures such as DNA-RNA hybrids. 2) The structure and properties of single and double stranded lesions in DNA resulting from chemistry initiated by hydrogen atom abstraction at C-4' will be established using a variety of methods including NMR spectroscopy. 3) The mutagenicity and genotoxicity of these lesions in E. coli will be investigated. 4) Isotope effects will be used as a probe to examine the mechanism of double stranded cleavage of DNA by BLM. 5) Physical biochemical methods will be used to examine the structure of activated BLM and to reexamine the kinetics of its formation and destruction. 6) The mechanism of ene diyne antibiotics will be examined using isotope effect methods in conjunction with physical biochemical methods including stopped flow visible spectroscopy. These studies are designed to expand the detailed mechanistic insight that has been achieved through this research project into the broader issues of molecular recognition and biological effects of these important chemotherapeutic agents.
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