Isotope effects on the sequence-specific cleavage of dC in d(AGC) sequences by neocarzinostatin: elucidation of chemistry of minor lesions

Kappen, Lizzy S.; Goldberg, Irving H.; Wu, Shao-Guo; Stubbe, JoAnne; Worth, Leroy; Kozarich, John W.

doi:10.1021/ja00163a049

Cited by 58 publications

(39 citation statements)

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“…Since the drug reaction was carried out under one-hit kinetic conditions, by quantitating the breakage at the I residue of the 1H and 2H substrate by densitometry, it is possible to calculate a small isotope selection effect (klH/k2H = 1.25). This value is in agreement with similar calculations at I residues at which 5' abstraction chemistry occurs (20,21) and is considerably smaller than that at C-1' of C (klH/k2H = 4.0) (17).…”

Section: Resultssupporting

confidence: 91%

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.

Meschwitz

Goldberg

1991

Proc. Natl. Acad. Sci. U.S.A.

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Use has been made of the mechanism of DNA deoxyribose damage by the ene-diyne-containing chromophore of the antitumor antibiotic neocarzinostatin to provide chemical evidence for the structure of the activated drug-DNA complex. Radical centers at C-2 and C-6 of the diradical form of the glutathione-activated chromophore abstract hydrogen atoms from C-1' of the C residue and C-5' of the I residue in AGC-GCT to generate a bistranded lesion consisting of an abasic site at C and a strand break at T. This laboratory has proposed a molecular model for the drug-DNA interaction in which the naphthoate moiety of the chromophore intercalates between Ar and G-C, placing the diradical core in the minor groove, so that the radical centers at C-6 and C-2 are close to C-5' of I and C-1' of C, respectively. To determine'which radical center abstracts one of the hydrogen atoms from C-5', the self-complementary oligodeoxynucleotide GCAGCGCTGC was synthesized with 2H at both 5' positions of thelT residue and treated with glutathione-activated chromophore. Sequencinggel electrophoresis showed that drug attack was limited to the I and C residues and that abstraction of2H from C-5' exhibited a small isotope selection effect of 1.25. 'H NMR spectroscopic examination of the reacted chromophore, isolated by HPLC, indicated that 2H was selectively abstracted by C-6, providing experimental corroboration of the model and further elucidating the chemical mechanism. Since direct strand breakage at the I residue exceeds (44% more) abasic site formation at the C residue, other models of drug-DNA interaction leading to only single-strand breaks are also considered.The DNA-damaging antitumor antibiotic neocarzinostatin (NCS) uniquely consists of a biologically active highly strained diacetylene-containing chromophore noncovalently complexed with its carrier apoprotein (for review, see ref. 1). The chromophore (2) structure is comprised ofthree subunits (3-5): a 5-methyl-7-methoxynaphthoate and a 2,6-dideoxy-2-(methylamino)galactose interlinked by a C12 subunit containing a bicyclo[7.3.0]dodecadiyne system bearing an epoxide and a cyclic carbonate moiety (Fig.

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Section: Resultssupporting

confidence: 91%

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.

Meschwitz

Goldberg

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…While C4' chemistry has been well defined, the question arises as to whether additional 'minor' chemistries can occur in the degradation of BLM by DNA. Our recent studies of the mechanism of ene diyne DNA cleavers using specifically isotopically labeled DNAs have revealed chemistry of minor lesions, not easily detectable by normal analytical methods (16)(17)(18). For example, the major lesion resulting from the interaction of neocarzinostatin (NCS) with DNA has been shown by Goldberg and his collaborators to occur at the 5' position of Ts and dAs (19).…”

Section: Introductionmentioning

confidence: 99%

“…For example, the major lesion resulting from the interaction of neocarzinostatin (NCS) with DNA has been shown by Goldberg and his collaborators to occur at the 5' position of Ts and dAs (19). However, chemistry at minor lesions such as C-i' at the dC in d(AGC) sequences (16) and C4' at the T and d(GT) steps were easily detected, using the isotopic effect methodology we have developed (17,18). Thus, chemistry of minor lesions, difficult to detect due to the unavailability of good analytical methods, deserves careful scrutiny as these minor lesions may play a significant role in the observed cytotoxicity.…”

Section: Introductionmentioning

confidence: 99%

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C–1′ chemistry

Absalon¹,

Krishnamoorthy²,

McGall³

et al. 1992

Nucl Acids Res

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Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.

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“…While these latter results imply that adduct formation may not involve C-1′ hydrogen abstraction, it must be noted that deuterium isotope selection effects have not been found in hydrogen abstraction reactions by certain other enediyne antibiotics, such as calicheamicin (32) and esperamicin (33), whereas they have been with NCS (34,35) and C-1027 (36). Further, such isotope effects have been founded to vary substantially with the sequence adjacent to the target site (34,35). Also, to realize a kinetic isotope effect, it is necessary that activated-drug binding has a significant off-rate component.…”

Section: Discussionmentioning

confidence: 96%

“…A substantial isotope effect (k H /k D ) 3.2) has been found in the NCS-induced formation of strand breakage and abasic sites at this nucleotide (as well as at U8) in the fully complementary hybrid substrate (19). While these latter results imply that adduct formation may not involve C-1′ hydrogen abstraction, it must be noted that deuterium isotope selection effects have not been found in hydrogen abstraction reactions by certain other enediyne antibiotics, such as calicheamicin (32) and esperamicin (33), whereas they have been with NCS (34,35) and C-1027 (36). Further, such isotope effects have been founded to vary substantially with the sequence adjacent to the target site (34,35).…”

Section: Discussionmentioning

confidence: 98%

Characterization of a Novel Covalent Monoadduct on the RNA Overhang of an RNA−DNA Hybrid Induced by Antitumor Antibiotic Neocarzinostatin

Zheng

Liu

et al. 1998

Biochemistry

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Neocarzinostatin chromophore (NCS-chrom) has been found previously to induce thiol-dependent abasic site formation and cleavage on the RNA strand of RNA-DNA hybrids. When a four-base nucleotide was deleted from the 5' end of the DNA strand, however, an unexpected product with slow mobility, potentially a drug adduct or interstrand cross-linked material, was observed instead. End-labeling of the RNA and DNA strands showed that only the RNA strand is present in the product. The product, isolated from a denaturing polyacrylamide gel, has a fluorescence spectrum similar to that of activated NCS-chrom, and thiols with different charges give rise to products with various mobilities on the gel, indicating that the product consists of a drug adduct with NCS-chrom and thiol attached to the RNA strand of the hybrid. Mass spectroscopic (MS) analysis shows that it is a monoadduct. Chemical sequencing of 5'- and 3'-end-labeled monoadduct and MS/MS data show that drug is attached only to the U9 of the RNA in the hybrid 5'-r(CACAGAAUU9CG)/5'-d(TTCTGTG). Unlike most other DNA adducts and cross-linked products found previously, this adduct can be formed readily in the presence of atmospheric oxygen. 2'-O-Methyl and 2'-H substitutions and nucleotide replacements on the 4-base RNA overhang affect monoadduct formation to different extents, reflecting the existence of a tight three-dimensional binding pocket for the activated drug. Stability of drug adduct to heat and aniline-HOAc treatment suggest that the drug is covalently bound to the ribose of U9. Since no deuterium isotope selection effect was observed for C-1' of U9, it is possible, although not required, that the drug abstracts H from another hydrogen source on the sugar of the targeted ribonucleotide.

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Isotope effects on the sequence-specific cleavage of dC in d(AGC) sequences by neocarzinostatin: elucidation of chemistry of minor lesions

Cited by 58 publications

References 0 publications

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.

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.

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C–1′ chemistry

Characterization of a Novel Covalent Monoadduct on the RNA Overhang of an RNA−DNA Hybrid Induced by Antitumor Antibiotic Neocarzinostatin

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