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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Metallobiochemistry Study Section (BMT)
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Massachusetts Institute of Technology
Schools of Arts and Sciences
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Chen, Jingyang; Ghorai, Manas K; Kenney, Grace et al. (2008) Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage. Nucleic Acids Res 36:3781-90
Chen, Jingyang; Dupradeau, Francois-Yves; Case, David A et al. (2008) DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics. Nucleic Acids Res 36:253-62
Chen, Jingyang; Dupradeau, Francois-Yves; Case, David A et al. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites. Biochemistry 46:3096-107
Chen, Jingyang; Stubbe, JoAnne (2004) Bleomycins: new methods will allow reinvestigation of old issues. Curr Opin Chem Biol 8:175-81
Chen, Jingyang; Stubbe, JoAnne (2004) Synthesis and characterization of oligonucleotides containing a 4'-keto abasic site. Biochemistry 43:5278-86
Junker, Hans-Dieter; Hoehn, Silvia T; Bunt, Richard C et al. (2002) Synthesis, characterization and solution structure of tethered oligonucleotides containing an internal 3'-phosphoglycolate, 5'-phosphate gapped lesion. Nucleic Acids Res 30:5497-508
Wu, Wei; Vanderwall, Dana E; Turner, Christopher J et al. (2002) Solution structure of the hydroperoxide of Co(III) phleomycin complexed with d(CCAGGCCTGG)2: evidence for binding by partial intercalation. Nucleic Acids Res 30:4881-91
Hoehn, S T; Turner, C J; Stubbe, J (2001) Solution structure of an oligonucleotide containing an abasic site: evidence for an unusual deoxyribose conformation. Nucleic Acids Res 29:3413-23
Hoehn, S T; Junker, H D; Bunt, R C et al. (2001) Solution structure of Co(III)-bleomycin-OOH bound to a phosphoglycolate lesion containing oligonucleotide: implications for bleomycin-induced double-strand DNA cleavage. Biochemistry 40:5894-905
Harsch, A; Marzilli, L A; Bunt, R C et al. (2000) Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis. Nucleic Acids Res 28:1978-85

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