Chemistry of the alkali-labile lesion formed from iron(II) bleomycin and d(CGCTTTAAAGCG)

Sugiyama, Hiroshi; Xu, Cheng; Murugesan, N.; Hecht, Sidney M.; Marel, Gijs A. van der; Boom, Jacques H. van

doi:10.1021/bi00401a011

Cited by 85 publications

(79 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In earlier experiments, this mobility has been found to be characteristic of a DNA fragment with a 3'-phosphoglycolate end ("glycolate") resulting from 4' chemistry (5,16,17). To determine the extent of the putative 4' chemistry, the drug reaction products were treated with hydrazine, which is known to react with the 4'-hydroxylated abasic site generated by bleomycin (18,19) and NCS-Chrom (5,6,20) to form a 3'-pyridazine derivative ("pyridazine") with elimination of the DNA fragment with a 3'-phosphate end (Scheme I). The pyridazine derivative (dashed arrow) migrates more slowly than the fragment with the 3'-phosphate end (Fig.…”

Section: Resultsmentioning

confidence: 99%

Neocarzinostatin acts as a sensitive probe of DNA microheterogeneity: switching of chemistry from C-1' to C-4' by a G.T mismatch 5' to the site of DNA damage.

Kappen

Goldberg

1992

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

View full text Add to dashboard Cite

show abstract

Section: Resultsmentioning

confidence: 99%

Neocarzinostatin acts as a sensitive probe of DNA microheterogeneity: switching of chemistry from C-1' to C-4' by a G.T mismatch 5' to the site of DNA damage.

Kappen

Goldberg

1992

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

View full text Add to dashboard Cite

show abstract

“…NCS was added to a final concentration of 10 nM to start the reaction, which was allowed to proceed for 1.5 h at 0 "C. This concentration of NCS is well below that which results in "one-hit" kinetics. To cleave abasic sites, and thus to express them as strand breaks, either hydrazine (1 M, pH 8), for 4'-hydroxylated abasic sites (Sugiyama et al, 1988;Kappen et al, 1991), or putrescine (1 M, pH 8), for all abasic sites (Lindahl & Andersson, 1972;, was added to a final concentration of 100 mM and the solution was incubated at room temperature or 37 OC, respectively, for 1 h. The topologic forms in 0.2 pg of drug-treated plasmid DNA were resolved on a 1% agarose gel. Densitometry of the negative images of the ethidium bromide-stained gel (LKB Ultrascan XL) showed linear variation of signal with DNA concentration over the DNA range studied (Dedon & Goldberg, 1990).…”

Section: Methodsmentioning

confidence: 99%

“…Cl'-hydrogen atom abstraction ultimately yields a putrescinecleavable 2'-deoxyribonolactone abasic site (Kappen & Goldberg, 1989), while C4'-chemistry partitions to form either a strand break with 3'-phosphoglycolate-and S-phosphateended fragments or a 4'-hydroxylated abasic site (Scheme I of Saito et al, 1989;Frank et al, 1991;Kappen et al, 1991). The latter lesion is cleaved by putrescine to form phosphate-ended fragments (Lindahl & Andersson, 1972; or by hydrazine to form a 3'-phosphopyridizine derivative (Sugiyama et al, 1988; 'Based on the proposal of Myers (1987). et al, 1991).…”

mentioning

confidence: 99%

Influence of thiol structure on neocarzinostatin activation and expression of DNA damage

Dedon¹,

Goldberg²

1992

Biochemistry

View full text Add to dashboard Cite

Neocarzinostatin (NCS) is an enediyne antitumor antibiotic that cleaves DNA following a thiol-induced electronic rearrangement to a diradical form. Structure-function studies with 11 thiol-containing compounds were undertaken to clarify the role of the thiol in NCS-mediated DNA damage. The rates of activation of NCS in the presence of DNA with the various thiols approximated a Brønsted relation (beta = 0.43, r2 = 0.86), which suggests that the basicity/nucleophilicity of the thiol is important to NCS activation. However, an additional contribution to NCS activation may arise from the affinity of the thiol for DNA, since there is a correlation between the concentration of thiol producing maximal DNA damage, assessed by quantitating the topologic forms of plasmid pBR322 following treatment with NCS, and the apparent ability of the thiol to bind to DNA by hydrophobic or electrostatic interactions. The overall second-order rate constants for the activation of NCS were found to be inversely correlated with the thiol optima; a plot of the former versus the reciprocal of the optimal thiol concentration revealed a first-order rate constant of activation of 0.013 s-1 in the presence of DNA. This indicates that maximal DNA damage occurs when NCS is activated with a half-life of 52 s, a relatively slow rate of activation that suggests that NCS binds to DNA before undergoing activation by thiol. Finally, an analysis of strand breaks in pBR322 shows that thiols possessing a carboxylate moiety produce larger quantities of bistranded DNA lesions than their esterified or non-carboxylate-containing counterparts.

show abstract

“…After incubation for 30 min at 370C, samples were passed over CM-25 Sephadex minicolumns (6) to remove bleomycin, split into two or three aliquots, and incubated for 1 hr at 370C in the presence or absence of putrescine (20 mM) or endonuclease III (8 units/ ml), in the same buffer but with 1 mM 2-mercaptoethanol. In some experiments, an aliquot was treated with hydrazine (20 mM) for 1 hr at 220C (7).…”

mentioning

confidence: 99%

Bleomycin-induced DNA lesions at mutational hot spots: implications for the mechanism of double-strand cleavage.

Steighner

Povirk

1990

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

143

135

View full text Add to dashboard Cite

Using various end-labeled, defined-sequence DNA substrates, we examined bleomycin-induced damage at several G'C base pairs which correspond to mutational hot spots. The most frequent lesions detected were single-strand breaks and single apu inic/apyrimidinic (AP) sites at the C residue, suggesting that this was the primary site of damage. Strand breaks and AP sites also occurred, but less frequently, at a secondary damage site-i.e., the directly opposed G residue in the complementary strand. However, damage at the secondary site occurred only when a strand break was present at the primary site, and AP sites at the primary site were never accompanied by closely opposed damage in the complementary strand. Thus, formation of a strand break at the primary damage site was a necessary though not sufficient condition for attack at the secondary site. Similar patterns were seen at other sequences attacked by bleomycin, although primary and secondary sites were sometimes staggered by one nucleotide position rather than directly opposed. These and other results suggest a mechanism of double-strand cleavage in which bleomycin Is reactivated during formation of the first strand break, and the reactivated drug subsequently attacks the complementary strand at a specific position which is not normally a site of bleomycin-induced cleavage. Regeneration of activated bleomycin could result from a reaction between Fe(w)'bleomycin and a 4'-peroxyl derivative of deoxyribose, both produced during formation of the strand break.The fact that bleomycin produces double-strand breaks with single-hit kinetics suggests that these lesions result from a single interaction between bleomycin and DNA (1, 2). However, the chemistry of activated bleomycin (3,4) gives no suggestion of bifunctionality, and the mechanism of doublestrand cleavage has remained uncertain. We recently showed that most bleomycin-induced double-strand breaks consist of a "primary" site, which follows the normal G-Y (Y =pyrim-idine nucleoside) specificity characteristic of bleomycininduced single-strand cleavage, and a secondary site (in the opposite strand), which is seldom a G-X sequence and is usually a site where single-strand cleavage is rare (2). In addition to double-strand breaks, bleomycin induces formation of apurinic/apyrimidinic (AP) sites with closely opposed strand breaks, and these bivalent lesions have been implicated in bleomycin-induced mutagenesis in A phage (5, 6). To clarify the mechanism by which bleomycin effects concomitant damage to both DNA strands, we have examined the detailed structure of bivalent lesions induced by bleomycin at specific sites in defined-sequence DNA substrates, particularly those corresponding to mutational hot spots.MATERIALS AND METHODS Materials. Recombinant plasmids were constructed by cloning either a synthetic 25-mer (pc1245, Fig. 1 Analysis of DNA Cleavage. Nondenaturing and denaturing gel electrophoresis and elution of fragments from gels have been described (2). Except in experiments employing hydrazine, all e...

show abstract

Chemistry of the alkali-labile lesion formed from iron(II) bleomycin and d(CGCTTTAAAGCG)

Cited by 85 publications

References 41 publications

Neocarzinostatin acts as a sensitive probe of DNA microheterogeneity: switching of chemistry from C-1' to C-4' by a G.T mismatch 5' to the site of DNA damage.

Neocarzinostatin acts as a sensitive probe of DNA microheterogeneity: switching of chemistry from C-1' to C-4' by a G.T mismatch 5' to the site of DNA damage.

Influence of thiol structure on neocarzinostatin activation and expression of DNA damage

Bleomycin-induced DNA lesions at mutational hot spots: implications for the mechanism of double-strand cleavage.

Contact Info

Product

Resources

About