This proposal is to continue studies to elucidate the detailed molecular mechanisms by which certain antitumor antibiotics damage DNA and interfere with DNA function. Not only will this work contribute to our understanding of how anti-cancer agents work, but should lead to the development of drugs with increased selectivity for neoplastic cells and lowered toxicity for the host. Important byproducts will be the uncovering of novel mechanisms of DNA damage and repair and of mutagenesis, and the development of new probes and tools for the study of gene structure and regulation. Members of a family of radiomimetic protein antibiotics, in particular neocarzinostatin (NCS), will be the focus of this investigation. Earlier work has shown that NCS contains a labile nonprotein chromophore of unique structure that possesses all the biological activity, binds to DNA by intercalation, and damages DNA when activated by thiol by selectively attacking C-5' of deoxyribose of mainly thymine residues to produce, in the presence of O2, strand breaks with nucleoside 5'-aldehyde at the 5'-end and phosphoryl at the 3'-end, and base release with the formation of apurinic/apyrimidinic sites, and in the absence of O2 covalent NCS chromophore- deoxyribose adducts. Evidence that a similar mechanism occurs in vivo will be sought by isolating DNA from NCS-treated mammalian cells and analyzing it by various techniques for the presence of the lesions found in vitro. Studies will also be performed 1) to determine the role of microheterogeneity of DNA structure in base sequence-dependent binding of NCS, using chemically synthesized oligodeoxynucleotides of defined sequence, 2) to determine structure-function relationships in the NCS chromophore by chemical modifications, 3) to obtain more detailed information on the molecular mechanism of DNA damage by chemical characterization of damage products (deoxyribose fragments, NCS chromophore-deoxyribose adducts, spent NCS chromophore, etc.), 4) to clarify how nitroaromatic radiation sensitizers, such as misonidazole, substitute for O2 in producing DNA damage, and 5) to characterize possible new enzymatic mechanisms of repair of NCS-induced DNA damage. These studies will be extended to include other related protein antibiotics, notably auromomycin, that have different attack site specificities and produce different DNA damage products.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM012573-24
Application #
3268384
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1977-01-01
Project End
1991-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
24
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Dedon, P C; Goldberg, I H (1992) Influence of thiol structure on neocarzinostatin activation and expression of DNA damage. Biochemistry 31:1909-17
Dedon, P C; Jiang, Z W; Goldberg, I H (1992) Neocarzinostatin-mediated DNA damage in a model AGT.ACT site: mechanistic studies of thiol-sensitive partitioning of C4' DNA damage products. Biochemistry 31:1917-27
Kappen, L S; Goldberg, I H; Frank, B L et al. (1991) Neocarzinostatin-induced hydrogen atom abstraction from C-4' and C-5' of the T residue at a d(GT) step in oligonucleotides: shuttling between deoxyribose attack sites based on isotope selection effects. Biochemistry 30:2034-42
Meschwitz, S M; Goldberg, I H (1991) Selective abstraction of 2H from C-5' of thymidylate in an oligodeoxynucleotide by the radical center at C-6 of the diradical species of neocarzinostatin: chemical evidence for the structure of the activated drug-DNA complex. Proc Natl Acad Sci U S A 88:3047-51
Galat, A; Goldberg, I H (1990) Molecular models of neocarzinostatin damage of DNA: analysis of sequence dependence in 5'GAGCG:5'CGCTC. Nucleic Acids Res 18:2093-9
Dedon, P C; Goldberg, I H (1990) Sequence-specific double-strand breakage of DNA by neocarzinostatin involves different chemical mechanisms within a staggered cleavage site. J Biol Chem 265:14713-6
Lee, S H; Thivierge, J O; Goldberg, I H (1989) DNA microstructural requirements for neocarzinostatin chromophore-induced direct strand cleavage. Nucleic Acids Res 17:5809-25
Kappen, L S; Lee, T R; Yang, C C et al. (1989) Oxygen transfer from the nitro group of a nitroaromatic radiosensitizer to a DNA sugar damage product. Biochemistry 28:4540-2
Galat, A (1989) A procedure for analysis of densitometric spectra. Electrophoresis 10:659-67
Galat, A (1989) CORGEN: a FORTRAN-77 generator of standard and non-standard DNA helices from the sequence. Comput Appl Biosci 5:279-86

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