STRUCTURE AND MECHANISM OF DNA INTERSTRAND CROSS-LINKING

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Many antitumor agents and toxic substances, including important environmental pollutants, exert their toxic effects by chemical reaction with DNA. In numerous cases, the regions of DNA which react with these substances and the structures of the resulting products are unknown. Understanding these details is critical to understanding the toxic effects of these substances. The proposed research will examine issues of chemical and biochemical significance arising from the interaction of antitumor agents, toxins, and toxicants with nucleic acids. The specific aims include: (1) We will evaluate the sequence specificity of cross-linking and solution structure of cross-linked DNAs derived from DNA interstrand cross-linking agents synthesized during the previous grant period which were designed to recognize and cross-link sequences up to 6 nucleotides in length. (2) We will quantify the sequence specificity, yield, and structures of the dG-to-dG intrastrand cross-links caused by mechlorethamine in duplex DNA, probe the origin of the unexpected interstrand cross- linking sequence specificity of mechlorethamine by studying a structural analog which extends the distance between the electrophiles, survey the efficiency and sequence specificity for interstrand cross- linking of DNA with other nitrogen mustards, and evaluate the impact of the mechlorethamine DNA-DNA interstrand cross-link on the affinity of proteins which recognize bent DNA in collaboration with Professor Essigmann. (3) We will define the covalent structure of the dG-to-dG cisplatin induced DNA-DNA interstrand cross-link at 5'-d(Gc) and determine the affinity of a 100-mer duplex containing this cross-link at a defined site for an HMG-family protein in collaboration with Professor Essigmann. (4) We will establish the DNA-DNA interstrand cross-linking sequence specificity and covalent structure of the nitrosourea-, chloroacetaldehyde-, and chromium(Vl)-induced interstrand cross-links. (5) We will determine whether RNA-DNA hybrid duplexes can be cross- linked by any of seven bifunctional electrophiles. These results could contribute background information necessary for the eventual rational design of agents targeted to react covalently with the RNA-DNA duplex.