This proposal seeks continued support for our studies directed toward the elucidation of the molecular mechanisms of DNA degradation by a variety of metal complexes, in particular the metallobleomycins (BLM). During the next funding period, we plan a comprehensive program which will include the following studies: 1. A complete analysis of the kinetic isotope effects on 4'- hydrogen abstraction by BLMs. We have designed methods to permit an accurate determination of these effects at individual sequence sites in a defined pBR322 fragment. This will allow us to study the potential effects of local conformation on the chemistry of the DNA-BLM interaction. 2. Continued investigations on the mechanism of deoxyribose ring fragmentation by Fe.BLM using 180 analysis. Our recent work has established that 180 incorporation into the phosphogycolate moiety is derived from the second 02 required for base propenal formation. Further studies are planned that will discriminate among several proposed pathways leading to cleavage. 3. The chemistry of BLM action on other DNA forms. Evidence suggests that BLM can react with A-form DNA. Model analysis has revealed that the chemistry of this process maydiffer from B DNA due to an altered accessibility of sugar hydrogens. Using techniques which we have developed, we will determine the mechanism of cleavage in appropriate model DNA's and DNA- RNA hybrids. Z DNA will also be studied. 4. The chemistry of other metallobleomycins. We plan to investigate the cleavage of DNA by cobalt-BLM and manganese- BLM. These complexes exhibit different activational requirements than the iron complex and yield different products. 5. The mode of BLM binding to DNA. The question of an intercalative vs. nonintercalative process for BLM binding remains open. Studies are planned to distinguish between these possibilities. 6. Model studies with other DNA cleaving agents. The probes which we have developed should be valuable in the determination of the mechanistic features of other agents which cleave DNA by hydrogen abstraction mechanisms. Ruthenium and cobalt complexes of 4,7-diphenylphenanthroline, methidium propyl- EDTA-Fe(II), and the 1,10-phenanthroline-copper complex are recent literature examples which are amenable to analysis by our technology.
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