Bleomycins (BLMs) are natural products that, in the presence of their required cofactors iron and oxygen, can catalyze cleavage of nucleic acids. These compounds are used clinically, usually in combination chemotherapy, and their mode of cytotoxicity is thought to be related to their ability to mediate double strand (ds)-DNA cleavage. Our structural studies on HOO-FeBLM, a putative analog of """"""""activated BLM"""""""" (HOO-Fe), bound sequence-specifically to oligonucleotides, have resulted in a model for how a single BLM molecule can effect ds-cleavage. We will continue to test our hypothesis of the role of the bithiazole tail and its partial intercalation in reorganization of the FeBLM from the primary to the secondary site of cleavage. A """"""""hot spot"""""""" for blunt-ended cleavage has been identified and the structure of HOO-FeBLM bound to the intact oligonucleotide and the oligonucleotide with a 3'-phosphoglycolate/5'-phosphate lesion at the primary site of cleavage will be determined by 20 NMR methods. The mode of binding relative to that predicted by our model will be established. BLMs tethered to cyclodextrins will be prepared and their ability to effect ds-cleavage using the hairpin methodology we developed will be investigated. Our model predicts that cyclodextrin should prevent intercalation and hence ds-cleavage. The rules to define DNA binding by examining all five (5) base-pair sequence space will be determined using a high throughput screen monitoring fluorescence changes in vitro. The relationship between binding and ds-cleavage will be examined. The relevance of our in vitro studies to ds-cleavage events in vivo will be examined with BLM, deglycoBLM and PLM which in vitro mediate distinct ratios of ss:ds cleavage. The BLM analogs will be radiolabeled and their uptake into cells examined and the ss:ds cleavage ratios determined in these cells. BLMs induce DNA repair enzymes such as human Apel. Methods to synthesize 4'-ketoabasic sites as well as ds-lesions generated in the presence of BLM are presented. The structures of these lesions will be determined. The repair of these lesions by Apel will be investigated in an effort to determine its relevance to the non-homologous end joining ds-break repair.
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