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.
