The goals of the proposed studies are: (1) to identify the DNA targets of enediyne anticancer drugs in whole cells; and (2) to define the features of drug structure and genomic organization that govern the location of the DNA damage. We will study the double-strand DNA breaks produced in whole cells by the enediyne anticancer drugs calicheamicin g1I and esperamicins A1 and C, using ligation mediated PCR (LMPCR) and a novel strategy for isolating sites of DNA damage.
In specific aims of expanding scope, we will relate enediyne structure to the location and quantity of drug-induced DNA damage at all levels of genomic organization, from DNA sequence to nuclear location. These are significant studies since they: (1) provide important information for the design of DNA-damaging agents; (2) test the relevance of in vitro models of DNA target selection; and (3) broaden our understanding of target selection by other genotoxins and drugs.
Specific Aims : 1) Map enediyne-induced DNA damage in whole cells at single-nucleotide resolution. Enediyne-induced DNA damage will be mapped by LMPCR over several hundred base pairs in model gene systems, beginning with the human PGK1 gene. Modifications of the LMPCR linker will both simplify the technique and increase its sensitivity for enediyne-induced DNA damage. 2) Define the role of chromatin structures in enediyne target selection in vivo. The scope of the studies will be broadened by mapping enediyne-induced DNA damage in relation to nucleosomes and other chromatin structures over several thousand base pairs of the model gene systems. 3) Determine the effect of DNA supercoiling on enediyne target selection in vitro and in vivo. Based on preliminary results, the effects of supercoiling on the location, quantity and chemistry of enediyne-induced DNA damage will be studied in vitro and in vivo. 4) To identify general features of enediyne target selection in vivo. Finally, the frame of reference will be expanded to relate enediyne-induced DNA damage to nuclear architecture and higher order chromatin structures (e.g., nuclear matrix, DNase I hypersensitive sites, telomeres and DNA transcription). A novel method to label and isolate sites of enediyne-induced DNA damage has been developed for these studies.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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Lees, Robert
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Massachusetts Institute of Technology
Other Domestic Higher Education
United States
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Barth, Marita; Dederich, Debra; Dedon, Peter (2009) An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA. Biotechniques 47:633-5
Lopez-Larraza, D M; Moore Jr, K; Dedon, P C (2001) Thiols alter the partitioning of calicheamicin-induced deoxyribose 4'-oxidation reactions in the absence of DNA radical repair. Chem Res Toxicol 14:528-35
Liang, Q; Dedon, P C (2001) Cu(II)/H2O2-induced DNA damage is enhanced by packaging of DNA as a nucleosome. Chem Res Toxicol 14:416-22
Salzberg, A A; Dedon, P C (2000) DNA bending is a determinant of calicheamicin target recognition. Biochemistry 39:7605-12
Wu, J; Xu, J; Dedon, P C (1999) Modulation of enediyne-induced DNA damage by chromatin structures in transcriptionally active genes. Biochemistry 38:15641-6
Xu, J; Wu, J; Dedon, P C (1998) DNA damage produced by enediynes in the human phosphoglycerate kinase gene in vivo: esperamicin A1 as a nucleosome footprinting agent. Biochemistry 37:1890-7
Salzberg, A A; Dedon, P C (1997) An improved method for the rapid assessment of DNA bending by small molecules. J Biomol Struct Dyn 15:277-84
LaMarr, W A; Sandman, K M; Reeve, J N et al. (1997) Differential effects of DNA supercoiling on radical-mediated DNA strand breaks. Chem Res Toxicol 10:1118-22
LaMarr, W A; Sandman, K M; Reeve, J N et al. (1997) Large scale preparation of positively supercoiled DNA using the archaeal histone HMf. Nucleic Acids Res 25:1660-1
Mathur, P; Xu, J; Dedon, P C (1997) Cytosine methylation enhances DNA damage produced by groove binding and intercalating enediynes: studies with esperamicins A1 and C. Biochemistry 36:14868-73