Pyrimidine Bases In DNA Research Articles

DNA associated with the cell membrane is involved in the inhibition of the skin rejection response induced by infusions of photodamaged alloreactive cells that mediate rejection of skin allograft.

Cell membrane DNA (cmDNA) is a form of DNA located on the surface of human and murine T-cells. It has recently been characterized as a target for photomodification by 8-methoxypsoralen (8-MOP) and long-wave ultraviolet light (UV-A). Whereas 8-MOP itself is biologically inert, photoactivated 8-MOP is covalently bound to pyrimidine bases in DNA. We have investigated the possible involvement of cmDNA photomodification in the induction of the suppression of skin allograft rejection in BALB/c mice preimmunized with 8-MOP/UV-A photodamaged alloreactive cells which mediates this allograft rejection. This suppression is demonstrated by inhibition of delayed-type hypersensitivity (DTH) and mixed leukocyte culture (MLC) responses. Splenocytes from BALB/c mice undergoing rejection of CBA/j skin graft which contained an expanded population of the effector T lymphocytes that mediate the rejection were treated with DNAse to remove cmDNA before or after treatment with 8-MOP and UV-A prior to infusion into naive BALB/c recipients. Mice that received pretreated effector cells were tested for MLC responses to CBA/j or B10 alloantigens before and after the DTH response. The DTH response of all groups of pretreated BALB/c mice to the relevant alloantigen was specifically suppressed as compared with the response of control mice. However, adoptive transfer of the suppression of the DTH response was optimally demonstrable only in syngeneic recipients of cells from donor mice treated with photodamaged alloreactive cells. Also, splenocytes from BALB/c mice immunized with photodamaged alloreactive cells demonstrated highly significant hyporesponsiveness and suppression of the MLC response of naive mice to the relevant alloantigen in the case of the primary MLC response, and to both alloantigens in the secondary MLC response which was totally eliminated by prior pretreatment of these effector cells with DNAse. Therefore, it appears that the suppression of the DTH response can be induced by pretreatment of the effector cells with DNAse and/or 8-MOP and UV-A but is adoptively transferable optimally only from mice which are recipients of photodamaged alloreactive cells. Moreover, the effectiveness of this treatment is decreased by prior removal of cmDNA from these cells. The presence of cmDNA is necessary for induction of suppression of the primary and secondary MLC responses in mice treated with photodamaged cells of allograft rejection.

The folate cofactor of Escherichia coli DNA photolyase acts catalytically.

Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.

DNA structure studies by resonance Raman spectroscopy

The application of ultraviolet resonance Raman spectroscopy (RRS) in DNA structure studies is reviewed. In exciting the purine and pyrimidine bases of DNA in their allowed electronic transitions at wavelengths shorter than 300 nm, the resonance Raman effect offers a specific source of information at each level of complexity. For the nucleic bases and mononucleotides, it gives information on the excited state geometries of the molecules through their intensity excitation profiles. For oligo- and poly-nucleotides, the Raman hypo/hyperchromism observed under the resonance conditions is very sensitive to base-base stacking interactions: thus it can monitor slight changes in these interactions, due to changes in the polymer geometries. Lastly the resonance Raman effect provides structural information through vibrational couplings between different components of the macromolecular buildings. Thus resonance Raman spectroscopy is shown to be a powerful technique for studying DNA conformational changes and gives very promising results in the selective investigation of chromophores in such elaborate molecular complexes as nucleic bases within DNA duplexes.

Characterization of DNA adducts derived from (.+-.)-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene and (.+-.)-7-methyl-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene

Structural characterizations of the DNA adducts derived from reaction of the racemic bay region anti-diol epoxides of dibenz[a,j]anthracene and 7-methyldibenz[a,j]anthracene with calf thymus DNA are presented. Quantities of adducts necessary for spectroscopic characterization were obtained from reactions of the respective diol epoxides with individual deoxyribonucleotides. Both hydrocarbon diol epoxides showed similar adduct profiles upon reaction with calf thymus DNA in vitro which were composed mainly of three deoxyguanosine and four deoxyadenosine adducts. No significant modification of pyrimidine bases in DNA was detected with either of the diol epoxides. Approximately 3 times more deoxyguanosine than deoxyadenosine residues in the DNA were found to be modified by both diol epoxides. The DNA reactions showed very similar stereo- and enantioselectivities with both diol epoxides. The stereochemistries of addition of the purine bases to the diol epoxides were determined from analysis of the NMR spectra of individual adducts. The predominant adducts formed were products of trans addition of the exocyclic amino group of purines to the diol epoxides. The enantiomeric nature of the various adducts was determined from reaction of the individual deoxyribonucleotides with the pure (+)-anti-diol epoxide of dibenz[a,j]anthracene. The major deoxyguanosine and deoxyadenosine adducts from reactions with DNA were found to arise from the (+)-enantiomer of both hydrocarbon diol epoxides. The high reactivities of both diol epoxides (24-38%) with DNA in solution are consistent with the high tumor-initiating activity exhibited by the diol epoxide of dibenz[a,j]anthracene relative to the parent hydrocarbon.

Diverse effects of three furocoumarins on human lymphocyte proliferation

The biological effects of three furocoumarins on the proliferation of human normal peripheral blood lymphocytes have been investigated. Mitogen-stimulated human lymphocytes were assayed “in vitro” by measuring 3H-thymidine ( 3H-TdR) incorporation in the presence and in the absence of 15–30 μM 3-carbethoxypsoralen (3-CPs), trimethylangelicin (TMA) and psoralen (PSR) with and without UV-A irradiation (365 nm). The three furocoumarins differ in their ability to form mono- and bi-functional adducts with DNA pyrimidine bases and in producing reactive species of oxygen. At low furocoumarin doses and short times of UV-A irradiation (15–30 sec) used in the present study, 3-CPs did not affect 3H-TdR incorporation in PHA-stimulated human lymphocytes, TMA strongly inhibited 3H-TdR incorporation, while unexpectedly, PSR increased 3H-TdR incorporation in the absence of irradiation, likely acting, under these experimental conditions, as a co-mitogen.

Investigations of Zn(II) complexes with DNA/RNA bases by means of quantum chemical calculations

The interaction of Zn 2+ ions with the five purine and pyrimidine bases of DNA and RNA, i.e., adenine (Ade), cytosine (Cyt), guanine (Gua), thymine (Thy) and uracil (Ura) has been studied using ab initio SCFHF computations with minimal basis set. Energy optimized structures of the possible complexes of the metal ion with the bases and the corresponding interaction energies have been determined. Zn 2+ coordinating simultaneously to the carbonyl oxygen and to one nitrogen in guanine or cytosine gives rise to the highest values of binding energy, followed by the nitrogen sites of adenine and guanine. The least favoured complexes are those involving binding of Zn 2+ to oxygen sites of thymine and uracil. One exception in this series of relative interaction energies is the position O4 in thymine. Most of the resulting geometrical features are similar to those found in previous studies on complexes of the bases with alkali and alkaline earth metal ions. The electronic structure of the coordinated ligands was analysed to obtain a picture of the H-bonding ability between the molecules in the Watson-Crick base pairs AT and GC in the presence of zinc ions. The results show that for Zn 2+ bound to guanine via N3 and N7, the H-bonds in GC are stabilized and also when Zn 2+ is coordinated to O2 of thymine (uracil) the H-bonds in AT(U) are stabilized, whereas for N3 coordination in adenine the AT(U) base pair is destabilized. For N7 coordination in adenine, it has been assumed that the coordination does not significantly change the interaction between Ade and Thy (Ura).

C4-cyclodimers of psoralen engaging the 4',5'-double bond.

Abstract— By irradiating psoralen (the parent linear furocoumarin) in thin solid film two new fluorescent photodimers. compounds I and II, have been isolated and characterized. Unlike previously isolated dimers of furocoumarins which showed a ‘pyrone‐pyrone’ structure involving in the C4‐cycloaddition the 3,4‐double bond of the furocoumarinic moiety, the new dimers show respectively a ‘furan‐furan’ and a ‘furan‐pyrone’ structure, involving therefore the 4′,5′‐double bond. By analogy with the photocycloaddition reactions between furocoumarins and pyrimidine bases of DNA, in this case too the 4′,5′‐double bond of the furocoumarin can be involved. The experimental conditions, however, in which irradiation is carried out play an important role.

Comparison of synthetic psoralen derivatives and 8-MOP in the inhibition of lymphocyte proliferation.

Psoralens are a family of furocoumarins that naturally occur in nature in small amounts in a variety of common vegetables, including figs and limes, and in larger quantities in plants originally identified in the Nile valley and in India.' These molecules have the remarkable quality of being activated by ultraviolet A (UVA) from a biologically inert form to one capable of forming covalent bonds with the pyrimidine bases of DNA.

DNA ASSOCIATED WITH THE CELL MEMBRANE IS INVOLVED IN THE INHIBITION OF THE SKIN REJECTION RESPONSE INDUCED BY INFUSIONS OF PHOTODAMAGED ALLOREACTIVE CELLS THAT MEDIATE REJECTION OF SKIN ALLOGRAFT

AbstractCell membrane DNA (cmDNA) is a form of DNA located on the surface of human and murine T‐cells. It has recently been characterized as a target for photomodification by 8‐methoxypsoralen (8‐MOP) and long‐wave ultraviolet light (UV‐A). Whereas 8‐MOP itself is biologically inert, photoactivated 8‐MOP is covalently bound to pyrimidine bases in DNA. We have investigated the possible involvement of cmDN A photomodification in the induction of the suppression of skin allograft rejection in BALB/c mice preimmunized with 8‐MOP/UV‐A photodamaged alloreactive cells which mediates this allograft rejection. This suppression is demonstrated by inhibition of delayed‐type hypersensitivity (DTH) and mixed leukocyte culture (MLC) responses. Splenocytes from BALB/c mice undergoing rejection of CBA/j skin graft which contained an expanded population of the effector T lymphocytes that mediate the rejection were treated with DNAse to remove cmDNA before or after treatment with 8‐MOP and UV‐A prior to infusion into naive BALB/c recipients. Mice that received pretreated effector cells were tested for MLC responses to CBA/j or B10 alloantigens before and after the DTH response. The DTH response of all groups of pretreated BALB/c mice to the relevant alloantigen was specifically suppressed as compared with the response of control mice. However, adoptive transfer of the suppression of the DTH response was optimally demonstrable only in syngeneic recipients of cells from donor mice treated with photodamaged alloreactive cells. Also, splenocytes from BALB/c mice immunized with photodamaged alloreactive cells demonstrated highly significant hyporesponsiveness and suppression of the MLC response of naive mice to the relevant alloantigen in the case of the primary MLC response, and to both alloantigens in the secondary MLC response which was totally eliminated by prior pretreatment of these effector cells with DNAse. Therefore, it appears that the suppression of the DTH response can be induced by pretreatment of the effector cells with DNAse and/or 8‐MOP and UV‐A but is adoptively transferable optimally only from mice which are recipients of photodamaged alloreactive cells. Moreover, the effectiveness of this treatment is decreased by prior removal of cmDNA from these cells. The presence of cmDNA is necessary for induction of suppression of the primary and secondary MLC responses in mice treated with photodamaged cells of allograft rejection.

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [ 14C]deoxycytidine, [ 3H]deoxyuridine, and [ 3H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [ 14C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [ 3H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [ 14C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.

Chemical reaction of DNA with cross-linking agents: Influences of base permutation on chemical reactivity

Certain psoralens can photoreact with the pyrimidine bases of DNA and form interstrand cross-links. In this communication, theoretical mechanism of the photoreaction of DNA with psoralens in the presence of near-ultraviolet light (320–400 nm, u.v.-A) is diagrammatically and quantitatively presented. The number of the receptor binding sites, the theorized photoreaction sites, can be estimated from the base composition of DNA if the extent of the photoreactivity of cytosine and the possibly steric hindrance effects of DNA double helix in photoreactivity can be experimentally determined. The photoreactivity of DNA can be expressed by the number of psoralens photobound. It is predicted that the photoreactivity of DNA is less than, or at most equal to, the number of receptor binding sites depending on the number of cross-links formed during photoreaction. The number of cross-links formed is the consequence of the base permutation which determines the constitution of cross-linking sites, and of the manners of psoralens intercalated in the stacked DNA bases. In addition, the formation of the cross-linking sites is also influenced by the photoreactivity of cytosine which is presently not understood. It is likely that theoretical mechanism of the chemical reaction of DNA with other cross-linking agents can be similarly hypothesized.

KINETIC ANALYSIS OF THE PHOTOREACTION (365 nm) BETWEEN PSORALEN AND DNA

Abstract— Psoralen complexed with DNA under irradiation at 365 nm forms monofunctional and bifunctional adducts with pyrimidine bases of DNA. while the fraction of psoralen which remains free in the aqueous solution forms photodimer and photooxidation products. We havc studied the photoreaction betwecn psoralen and DNA from a kinetic point of view. Only some products of this photoreaction could be measured quantitatively. Thus. in order to present a picture of the overall photoreaction. we have represented this by a mathematical model consisting of a system of seven differential equations. Utilizing all the experimental data that it was possible to obtain. we were able to estimate rate constants for formation of all photoproducts. From the general picture thus obtained. we conclude that monofunctional adducts are formed in a four to one ratio to bifunctional adducts. Among the monofunctional photoproducts. 3,4‐cycloadducts occur with a higher yield than 4',5'‐cycloadducts. The combined rate constant of formation of photodimer and photooxidation products of psoralen is small. indicating that they are marginal by‐products relative to the cycloadducts formed in the photoreactions with DNA.

FLUORIMETRIC DETERMINATION OF 4‘,5’‐CYCLOADDUCTS IN THE DNA‐PSORALEN PHOTOREACTION

Abstract— 4′,5′‐Photocycloadducts of psoralen with pyrimidine bases of DNA have been quantitatively determined by fluorescence measurements on the acid hydrolysate of DNA‐psoralen photoadducts.

Photoproducts from DNA pyrimidine bases and polycyclic aromatic hydrocarbons.

The major photoproduct formed between benzo[a]pyrene and thymine is identified as 1-(benzo[a]pyren-6-yl)-thymine by means of spectroscopic analysis and isotopic syntheses. Irradiation of 1-methylcytosine hydrochloride and anthracene gives two isolable photoproducts of which one is assigned the structure 5-(anthracen-9-yl)-1-methylcytosine.

The Arecibo message of November, 1974

On November 16, 1974, the Arecibo Observatory transmitted at 2380 MHz at an effective bandwidth of 10 Hz a message directed at the globular cluster M13. The message consists of a 1679-bit picture portraying a counting scheme, five biologically significant atoms (H, C, O, N, and P), the genetic structure of the four purines and pyrimidine bases of DNA; a schematic of the DNA double helix with an order-of-magnitude estimate of the number of base pairs; a representation of a human being and his or her dimensions; a depiction of the solar system with an indication that human beings inhabit the third planet and an estimate of the human population of the Earth; and finally, a schematic representation of the Arecibo Observatory and a description of its dimensions.

Photoreactivity of hydroxypsoralens and their photobiological effects in Bacillus subtilis.

Abstract— Although psoralen and many substituted psoralens are potent skin‐photosensitizing agents, hydroxypsoralens are not. A satisfactory molecular interpretation of this structural specificity has been given in terms of dissociation of the hydroxyl group of 5‐ and 8‐hydroxypsoralens in their excited states. The dissociation process in the S1 state effectively competes with S1→T1 intersystem crossing, thus reducing the photoreactive T1 population. The T1 states of the anions are more delocalized than those of neutral psoralens so that they are less reactive toward photocycloaddition with pyrimidine bases of DNA. The lack of significant phosphorescence of hydroxypsoralens in ionizing solvent or in the presence of base at low temperatures (14–77 K.) indicates ineffective S1→T1 and/or effective T1→S0 intersystem crossing. These factors make hydroxypsoralens unreactive, electronically and kinetically, as skin photosensitizers, which are known to react with DNA. In correlation with the hydroxypsoralens' spectroscopic characterization, they are also found to be ineffective or less effective photosensitizers in Bacillus subtilis.

Proton magnetic resonance studies of ultraviolet-irradiated apurinic acid.

In apurinic acid, a single-stranded polydeoxyribonucleotide easily obtained upon depurination of DNA, the proton resonances arising from thymine and cytosine are readily observable in aqueous solution of 25 degrees C. Two methyl thymine resonances, centered at 1.88 ppm and separated by 0.045 ppm, are observed. We attribute the downfield methyl resonance to thymines with no pyrimidine nearest neighbors and the upfield methyl resonance to thymines having pyrimidine neighbors in the 3' and/or 5' positions. Upon ultraviolet irradiation, the upfield methyl and thymine H-6 resonances decrease in amplitude and two methyl resoances appear at 1.63 and 1.52 ppm, corresponding, respectively, to cytosine-thymine and thymine-thymine cyclobutane dimers. Photoreversal eliminates these two minor methyl resonances from the pmr spectrum. We conclude that apurinic acid provides a suitable model system for pmr studies of chemically modified pyrimidine bases in DNA.

MODE OF ACTION OF ETHYLENE OXIDE ON SPORES OF Clostridium botulinum 62A

SUMMARY— Death of spores of Clostridium botulinum when exposed to gaseous ethylene oxide followed first order kinetics. Supplementation of a synthetic medium with the purine and pyrimidine bases of DNA and RNA indicated, as judged by outgrowth from spores which had received sublethal ETO treatments, that the lethal action of ETO on the spores was through alkylation of the guanine and adenine components of DNA. Observed impairment of RNA and protein synthesis was considered an indirect effect resulting from alkylation of DNA components; however, additional evidence bearing on this point is needed to support a more definite conclusion.

Localization of fluorescein-labeled antinucleoside antibodies in glomeruli of patients with active systemic lupus erythematosus nephritis.

Renal tissues from two groups of patients were studied with fluorescein-labeled (Fl-) antibodies (Abs) to immunoglobulins, complement, and antibodies prepared in rabbits against BSA conjugate of 5-methyluridine (T) and cytidine (C), the latter two of which react specifically with denatured DNA. The first group consisted of 13 SLE patients, and the second consisted of 53 patients with non-SLE nephropathies. The data obtained from the two groups of patients were used for comparison, and they showed the following:(a) Fl-Abs to immunoglobulins and complement were bound in the glomeruli of tissues from all patients with active SLE glomerulonephritis characterized by deposits of foreign material in glomerular capillary walls (GCW). The fluorescent pattern was granular, corresponding to the distribution of the glomerular deposits, as seen by electron microscopy. Fl-Abs reactive with thymine and cytosine were bound in the GCW of eight of the nine patients with active SLE glomerulonephritis and showed the same granular distribution. The capacity of these latter Fl-Abs to stain the GCW was removed by absorption with the homologous antigen or denatured DNA.(b) Fl-Abs to immunoglobulins, complement, and pyrimidine bases of DNA did not react with the GCW of two SLE patients without clinical and histologic evidence of glomerulonephritis or with the sclerotic glomeruli of two uremic patients with chronic "burned out" lupus nephritis.(c) The glomeruli of 47 of the 53 patients with other nephropathies bound Fl-Abs to immunoglobulins and complement to some extent, and in 26, the localization appeared as marked as in the patients with active SLE glomerulonephritis. Fl-Abs reactive with thymine and cytosine were bound in the GCW of only one of the renal tissues from the 53 non-SLE patients. In the remaining 52, no binding was seen.(d) The findings are consistent with the hypothesis that antigen-antibody complexes, formed by denatured DNA, specific antibody, and complement, are present in the deposits of foreign material accumulated in the GCW of patients with active SLE glomerulonephritis, and that they may contribute to the pathogenesis of this renal disease.

Fundamental dimensions of the bases in nucleic acids

Revised dimensions, together with their standard errors, for the purine and pyrimidine bases of DNA and RNA have been derived from the results of 21 recent crystal structure determinations. These new dimensions are more precise than those which have been published previously. It is pointed out that the two purines do not have identical dimensions in their rings, nor do the two pyrimidines. It is also pointed out that in many of the pairs of external bond angles the two are not equal, contrary to previous assumptions.