The bleomycins are antitumor antibiotics that are used clinically in the treatment of head and neck and testicular cancer. Their cytotoxicity is thought to be related to their ability, in the presence of their required cofactors metal and O2, to bind to and degrade duplex DNA. They can catalyze both ss and ds cleavage events. The rarer and probably more cytotoxic ds-cleavage events are thought to be mediated by a single BLM which requires its reactivation by unprecedented chemistry and reorganization some 15 to 18A to the second strand without dissociating from the DNA. Recently the first model of a structure of a putative activated BLM analog, the hydroperoxide of CoBLM (HOO- CoBLM) has been elucidated by 2D NMR spectroscopic methods and molecular modeling. These studies provide an explanation for the ode of binding, the sequence specificity for pyrimidines (Py) in GpPy sequences and the chemical specificity for 4'-H atom abstraction of the deoxyribose moiety of the Py. The model of the structure raises questions that will be addressed in this proposal. Is this mode of binding generic or sequence specific? Is the HOO-CoBLM analog a good model of activated iron BLM (HOO- FeBLM)? It also provides a hypothesis for how a single BLM molecule can catalyze cleavage on both strands of the duplex in a blunt-ended or 5'-staggered fashion. This hypothesis points to the importance of the peptide linker connecting the metal binding domain and bithiazole tail. These questions will be examined using 2D NMR methods, technology using hairpin oligonucleotides and page to examine quantitatively ss:ds cleavage ratios, and a variety of BLM analogs synthesized in Boger~s~s~s lab. The BLMs generate two types of lesions in the DNA: 3'- phosphoglycolate ended and 4'-ketoabasic site lesions. Methods to synthesize these lesions using phosphoramidite chemistry are presented. These synthetic methods should allow their structural characterization in different sequence contexts using 2D NMR methods and analysis of their stability. The enzymology of repair of these lesions, by repair enzymes (Endo IV, Exo III, Apn1, ape 1) will also be examined. Finally the genotoxicity and mutagenicity of these two lesions in vivo in E. Coli will be examined. Resistance to BLM has limited its clinical usefulness. Studies will be undertaken to determine if the apo form of BLM or a metallo BLM are bound by the proteins associated with resistance and their relative affinities. Understanding resistance mechanisms can lead to rational removal of the resistance problem.
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