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C W Haidle

Publications and source records attributed to C W Haidle.

9 recordsLinked to original sources
PubMed1979

Site specificity of bleomycin-mediated single-strand scissions and alkali-labile damage in duplex DNA.

Form II PM2 DNA, which contained bleomycin-mediated single-strand breaks, was purified and treated with the extracellular endonuclease from Alteromonas BAL 31. This enzyme cleaves the phosphodiester backbone opposite a single-strand break to yield a double-strand break. The locations of these double-strand breaks were determined relative to the cleavage sites produced by the restriction enzyme HindIII. The experimental procedure was as follows. Form I PM2 DNA was treated with bleomycin to produce alkali-labile bonds. These were hydrolyzed by alkali treatment and the DNA, now containing single-strand breaks, was purified and treated with the BAL 31 enzyme and the HindIII enzyme to determine the positions of the original alkali-labile bonds. It was found that the single-strand breaks and alkali-labile bonds were introduced at preferred sites on the PM2 genome, since electrophoretic analyses of the DNA after the HindIII digestion revealed DNA bands of discrete sizes. The molecular weights of the DNA fragments produced by these treatments indicate that single-strand breaks and alkali-labile bonds occur at the same sites as those previously determined for direct double-strand scissions introduced by bleomycin at neutral pH. Some of the specific sites of double-strand scissions mediated by bleomycin at neutral pH (Lloyd et al., 1978b) are also shown here to be relatively more reactive than other sites when the DNA contains superhelical turns.

Bacteriophages↗
PubMed1979

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.

Bacteriophages↗
PubMed1979

Noncovalent intermolecular crosslinks are produced by bleomycin reaction with duplex DNA.

Reaction of covalently closed circular PM2 bacteriophage DNA with the anticancer drug bleomycin produces nicked circular (form II) and linear duplex (form III) DNA [Lloyd, R.S., Haidle, C.W. & Robberson, D.L. (1978) Biochemistry 17, 1890-1896]. As the reaction proceeds, the frequencies of both form II and form III DNA increase and, concomitantly, an increasing fraction of the DNA mass is found to be in crosslinked structures. Approximately 16% of the PM2 DNA mass is found to be crosslinked after 30 min of reaction with bleomycin at 0.5 microgram/ml. The proportion of each form found in any given crosslinked structure is directly related to the concentration of uncrosslinked (monomeric) forms. Multiple sites of crosslinking occur, and these frequently extend over a region of approximately 500 nucleotide pairs. The intermolecular crosslinked bonds are dissociated by extensive dialysis or by the addition of salt at high concentration (0.8 M NaCl), as would be expected if the bonds were noncovalent. Because intramolecular covalent crosslinks between complementary strands are not detected, it is suggested that intermolecular crosslinks are formed by noncovalent association of bleomycin molecules bound to each of the forms of DNA.

Bacteriophages↗
PubMed1978

Bleomycin-specific fragmentation of double-stranded DNA.

Brief exposure of covalently closed circular duplex PM2 DNA to low concentrations of the clinical bleomycin mixture (Blenoxane) resulted in specific fragmentation of the genome that does not depend on the presence of superhelical turns. The double-strand breaks are in fact produced at several discrete sites on the PM2 genome but frequently occurring near the HpaII restriction endonuclease cleavage site. Initial rates of formation of nicked circular and linear duplex PM2 DNAs are reduced to different extents as the ionic strength of the reaction is increased. Increasing ionic strength is most effective in reducing the initial rate and overall yield of apparent double-strand scissions compared with single-strand scissions in the bleomycin-treated PM2 DNA.

Bacteriophages↗
PubMed1977

Effect of bleomycin on the synthesis and function of RNA.

Bleomycin inhibits cellular RNA synthesis and the inhibition is nonspecific. The ratio of polyadenylate- [poly(A)] containing RNA to non-poly(A)-containing RNA in the drug-treated human lymphocytic cells, line Wil2, was the same as that in untreated cells. Poly(A) RNA isolated from untreated cells was used as a template for reverse transcriptase to synthesize complementary DNA, which was then used as a probe to assay the sequence diversity of poly(A)RNA's from treated and untreated cells. It was found that essentially all of the poly(A) RNA's in the untreated cells were also present in the treated cells. The effect of bleomycin on the biological activity of messenger RNA (mRNA) was tested with globin mRNA in a wheat germ embryo translation system. Although bleomycin inhibited protein synthesis at high concentrations, the inhibition was not due to a modification of mRNA. This was evidenced by the fact that no decrease in the ability of mRNA to function in the test system was found when globin mRNA was pretreated with high concentrations of bleomycin followed by removal of the drug.

Bleomycin↗