SPECIFICITY OF INTERCALATOR DIRECTED GENOTOXICITY

拟议研究的总体目标是确定下列机制

The broad goal of the proposed studies is to define mechanisms by which intercalating agents mediate mutation and lethality at the DNA level. This knowledge may ultimately be exploited to enhance the selective anti-tumor effect or reduce the non-specific genotoxicity of these agents. Recent work suggests that doxorubicin causes deletion mutations in the lacI gene of excision repair proficient and deficient strains of E. coli. Deletion endpoints occur adjacent to sites of apparent drug intercalation in the DNA. A focus of doxorubicin induced deletions in the lac operator suggests a drug specific interaction with this structure. The mechanisms mediating these mutations are not clear but may involve topoisomerase II and excision repair enzymes. The objective of this research project is to identify mechanisms by which intercalating agents mediate DNA damage in vivo. Specifically, the roles of topoisomerase II and excision repair enzymes in addition to sequence specific intercalator interactions with DNA will be addressed. Mutational spectra will be analyzed at the DNA sequence level in E. coli with actinomycin D which has a proven sequence specificity which differs from doxorubicin. Specific DNA sequences in lacI where doxorubicin and actinomycin D interact with DNA alone, topoisomerase II or excision repair enzymes will be defined in vitro. This will be done with DNase I footprinting, damage distribution and cleavable complex assays. The defined sequences will be incorporated into lac operator containing constructs for integration into the genome of mammalian cell lines. These construct containing cell lines will then comprise a stringent deletion detection system. Sequences which target in vitro effects of intercalating agents will be correlated with their ability to promote deletion in mammalian cells. This will allow identification of mechanisms leading to deletion in vivo.