Single molecules of double-stranded DNA (dsDNA) were stretched with force-measuring laser tweezers. Under a longitudinal stress of approximately 65 piconewtons (pN), dsDNA molecules in aqueous buffer undergo a highly cooperative transition into a stable form with 5.8 angstroms rise per base pair, that is, 70% longer than B form dsDNA. When the stress was relaxed below 65 pN, the molecules rapidly and reversibly contracted to their normal contour lengths. This transition was affected by changes in the ionic strength of the medium and the water activity or by cross-linking of the two strands of dsDNA. Individual molecules of single-stranded DNA were also stretched giving a persistence length of 7.5 angstroms and a stretch modulus of 800 pN. The overstretched form may play a significant role in the energetics of DNA recombination.

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Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules

The biochemistry and medical significance of the flavonoids.

We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.

Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

Flavonoids, a class of natural products of high pharmacological potency

extremely grateful to the Burroughs Wellcome Co. and to Hoffmann-La Roche Inc. for their support of my research in Tuckahoe from 1960 to 1970 and in Nutley from 1970 to the present. Looking back at the names of earlier recipients of the Clowes Award, I realize that I am the 6th Clowes Lecturer who, at one time or another in his career, has been associated with the McArdle Laboratory for Cancer Research at the University of Wisconsin. I believe that the large number of awardees associated with McArdle is telling us something about the genius of Dr. Harold Rusch who established McArdle with a handful of outstanding young investigators. Dr. Rusch helped to develop in his associates an intense devotion to fundamental cancer research, and he provided his colleagues with an at mosphere that encouraged them to have critical but construc tive interactions with each other in ways that stimulated excel lence in research by all of the members of the group. In 1952, I had the good fortune of coming to McArdle as a graduate student of Drs. James and Elizabeth Miller. Although I failed in my first research assignment, which was to synthesize pure 2amino-1-naphthol, the Millers had great patience with me, and they tried very hard to instill their high standards into my research. Whatever small accomplishments I may have made to biomÃ©dical research were in large part the result of the excellent training and encouragement that I received from Jim and Betty Miller. An important problem that has been of great interest to me for many years is individuality in the response of human beings and other living organisms to foreign chemicals. Why does a drug or an environmental pollutant cause toxicity in one person and not in another person? Why do some cigarette smokers develop lung cancer whereas other cigarette smokers do not? One of the causes of variability in the response of human beings to a foreign chemical is individuality in the rate of metabolism of the chemical. By the mid-1960s, it was recog nized by clinical pharmacologists that the plasma half-lives and steady state plasma concentrations of drugs varied by as much as 10to 20-fold among different individuals (42, 115, 204, 318). Because it seemed likely that there were also large differences in the metabolism of chemical carcinogens in dif ferent individuals, we investigated the metabolism of BP2 by

https://cancerres.aacrjournals.org/content/canres/42/12/4875.full.pdf

Induction of Microsomal Enzymes by Foreign Chemicals and Carcinogenesis by Polycyclic Aromatic Hydrocarbons: G. H. A. Clowes Memorial Lecture

BEFORE it can exert its mutagenic and carcinogenic activities1,2 benzo(a)pyrene (BP) requires metabolic activation and it is widely considered3–5 that the initiating event in these processes is covalent binding to DNA. The predominant form of the activated hydrocarbon is a diol epoxide6–11, of which four possible isomers are (+) and (−) enantiomers of 7β,8α-dihydroxy-9α-10α-epoxy-7,8,9,10-tetrahydro-BP (anti-BP diol epoxide) and (+) and (−) enantiomers of 7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro-BP (syn-BP diol epoxide). Enzymatic formation of the (+) anti and (+) syn configurations have been reported elsewhere12,13. We have shown that the racemic (±) anti-BP diol epoxide forms two stable covalent adducts each with the exocyclic amino groups of deoxyguanosine, deoxyadenosine and probably deoxycytidine14. We report here that we have now resolved the enantiomers of (±) anti-BP diol epoxide and reacted the optically pure hydrocarbons with DNA. Each enantiomer reacts with the exocyclic amino group of deoxyguanosine to form a pair of diasteriomers in a ratio which depends on the secondary structure of the nucleic acid. The (+) enantiomer reacts preferentially or asymmetrically with deoxyguanosine in double stranded DNA but the two enantiomers react equally with the same position in single-stranded DNA. The basis for the stereoselective binding is unknown but may involve formation of sterically orientated physical complexes before covalent attachment of the hydrocarbon to DNA. The mode of binding in the deoxyadenosine sites, on the other hand, must be different, as the (+) and (−) enantiomers react equally in either single- or double-stranded DNA.

Double-stranded DNA stereoselectively binds benzo( a )pyrene diol epoxides

The isolation and structural characterization are described of the major monoaddition products formed in the photoreaction of two naturally occurring psoralens, 8-methoxypsoralen and 4,5',8-trimethylpsoralen, with high molecular weight, double-stranded DNA. Hydrolysis of the psoralen-modified DNA and subsequent chromatography resulted in the isolation of four modified nucleosides from each psoralen. Structural characterization was accomplished by mass spectrometry and 1H NMR analysis. The major products, accounting for 44-52% of the covalently bound psoralen, are two diastereomeric thymidine adducts formed by cycloaddition between the 5,6 double bond of the pyrimidine and the 4',5' (furan) double bond of the psoralen. A minor product, less than 2% of the covalently bound psoralen, is a furan-side adduct to deoxyuridine, derived from an initially formed deoxycytidine adduct by hydrolytic deamination. A fourth product is a thymidine adduct where cycloaddition has taken place between the 5,6 double bond of the pyrimidine and the 3,4 (pyrone) double bond of the psoralen. This pyrone-side adduct accounts for 19% of the covalently bound 8-methoxypsoralen but for less than 3% of the covalently bound 4,5'8-trimethylpsoralen. All of the isolated adducts have cis-syn stereochemistry. The stereochemistry and product distribution of the adducts are determined in part by the constraints imposed by the DNA helix on the geometry of the noncovalent intercalation complex formed by psoralen and DNA prior to irradiation.

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Psoralen-deoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8-methoxypsoralen and 4,5'8-trimethylpsoralen.

Psoralen photochemistry is specific for nucleic acids and is better understood at the molecular level than are all other methods of chemical modification of nucleic acids. These compounds are used both for in vivo structure analysis and for photochemotherapy since they easily penetrate both cells and virus particles. Apparently, natural selection has selected for membrane and virus penetrability during the evolution of these natural products. Most cells are unaffected by relatively high concentrations of psoralens in the absence of ultraviolet light, and the metabolites of the psoralens have thus far not created a problem. Finally, psoralens form both monoadduct and cross-links in nucleic acid helices, the yield of each being easily controlled by the conditions used during the photochemistry.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0033583500005242

The reaction of the psoralens with deoxyribonucleic acid

The isolation and characterization of pyrimidine-psoralen-pyrimidine photodiadducts from DNA are reported for the first time. For each of the four psoralens studied, a single pair of diastereomeric thymidine-psoralen-thymidine photodiadducts, each with cis-syn stereochemistry, was found to account for > 90% of the diadducts formed. Additionally, pulse-chase experiments that establish that these photo cross-links are formed by cycloaddition of a second thymidine residue to the 3,4 double bond (pyrone side) of an initially formed 4',5' (furan-side) psoralen-thymidine photomonoadduct have been carried out.

/pdf/isolation-and-characterization-of-pyrimidine-psoralen-4n3p8hncqj.pdf

Isolation and characterization of pyrimidine-psoralen-pyrimidine photodiadducts from DNA

CHEMICAL carcinogens, for example, benzo[a]pyrene (BaP), are metabolised to reactive intermediates which bind covalently to cellular macromolecules1–3 and the extent of binding seems to correlate with the carcinogenic potency of the hydrocarbons1. Through various mutagenicity, metabolism and binding studies, the intermediate which undergoes formation of a stable covalent complex with DNA has been identified as 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene (BaP diol epoxide)4–9. The structure of a covalent adduct formed between this hydrocarbon and poly(G) has also been established6–9. We report here the identity of several adducts obtained by reacting (±)BaP diol epoxide with tritium-labelled DNA. Four differently labelled lots of DNA were synthesised in vitro with DNA polymerase I by incorporating in each case three unlabelled and one tritium labelled nucleoside triphosphate. Through the use of this unambiguous labelling technique we have demonstrated that activated BaP forms two adducts with deoxyguanosine, two with deoxyadenosine and possibly one wtih deoxycytidine, while reaction with deoxy-thymidine was not detected. This approach also allowed the relative percentage of each adduct to be calculated. The deoxyguanosine adducts predominated and constituted 92% of the total stable covalent adducts formed.

/pdf/benzo-a-pyrene-diol-epoxide-covalently-binds-to-3tbg71r9rc.pdf

Kenneth Straub

Papers

Double-stranded DNA stereoselectively binds benzo( a )pyrene diol epoxides

Psoralen-deoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8-methoxypsoralen and 4,5'8-trimethylpsoralen.

The reaction of the psoralens with deoxyribonucleic acid

Isolation and characterization of pyrimidine-psoralen-pyrimidine photodiadducts from DNA

Benzo[a]pyrene diol epoxide covalently binds to deoxyguanosine and deoxyadenosine in DNA