Photochemical demonstration of stacked C.C+ base pairs in a novel DNA secondary structure

The secondary structure of the alternating polydeoxynucleotide sequence poly[d(C-T)] was studied as a function of pH by ultraviolet absorbance and circular dichroism spectroscopy and by the analysis of UV-induced photoproducts. As the pH was lowered, poly[d(C-T)] underwent a conformational transition that was characterized by changes in the long-wavelength region (280-320 nm) of the CD spectrum. These changes have previously been interpreted as evidence for the formation of a core of stacked, protonated C X C+ base pairs in a double-helical complex of poly[d(C-T)], with the thymidyl residues being looped out into the solvent [Gray, D. M., Vaughan, M., Ratliff, R. L., & Hayes, F. N. (1980) Nucleic Acids Res. 8, 3695-3707]. In the present work, poly[d(C-T)] was labeled with [U-14C]cytosine and [methyl-3H]thymine and irradiated at pH values both above and below the conformational transition point (monitored by CD spectroscopy). The distribution of radioactivity in uracil means value of uracil dimers, uracil means value of thymine dimers (the deamination products of cytosine means value of cytosine and cytosine means value of thymine dimers, respectively), and thymine-means value of thymine dimers was then determined. As the pH was decreased, we found an increase in the yield of uracil means value of uracil dimers and a decrease in the yield of uracil means value of thymine dimers, which occurred concomitantly with the change in the CD spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytosine photoproduct-DNA glycosylase in Escherichia coli and cultured human cells

Ultraviolet irradiation of DNA produces a variety of pyrimidine base damages. The activities of Escherichia coli endonuclease III and a human lymphoblast endonuclease that incises ultraviolet-irradiated DNA at modified cytosine moieties were compared. Both the bacterial and human enzymes release this cytosine photoproduct as a free base. These glycosylase activities are linear with times of reaction, quantities of enzyme, and irradiation dosages of the substrates. Both enzyme activities are similarly inhibited by the addition of monovalent and divalent cations. Analysis by DNA sequencing identified loci of endonucleolytic incision as cytosines. These are neither cyclobutane pyrimidine dimers, 6-(1,2-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4(1H,3H)-pyrimidinediones, nor apyrimidinic sites. This cytosine photoproduct is separable from unmodified cytosine by high-performance liquid chromatography. This separation should facilitate identification of this modified cytosine and elucidation of its biological significance.     

Sequence specificity of cyclobutane pyrimidine dimers in DNA treated with solar (ultraviolet B) radiation.

Cyclobutane pyrimidine dimers were quantified at the sequence level after irradiation with solar ultraviolet (UVB) and nonsolar ultraviolet (UVC) light sources. The yield of photoproducts at specific sites was dependent on the nucleotide composition in and around the potential lesion as well as on the wavelength of ultraviolet light used to induce the damage. Induction was greater in the presence of 5' flanking pyrimidines than purines; 5' guanine inhibited induction more than adenine. UVB irradiation increased the induction of cyclobutane dimers containing cytosine relative to thymine homodimers. At the single UVC and UVB fluences used, the ratio of thymine homodimers (T mean value of T) to dimers containing cytosine (C mean value of T, T mean value of C, C mean value of C) was greater after UVC compared to UVB irradiation.     

The two-step model of UV mutagenesis reassessed: deamination of cytosine in cyclobutane dimers as the likely source of the mutations associated with photoreactivation

Summary A large increase in the incidence of bacteriophage mutants is found after photoreactivation of UV-irradiated phage S13. The increase was seen only when the irradiated phage were stored before they were photoreactivated; the maximum mutation frequency was achieved after storage for 2 h at 4° C or 30 min at 37° C. The mutations can be attributed entirely to deamination of cytosine in cyclobutane dimers. Naked S13 DNA was stored for 2 h at 37° C after being irradiated with wavelengths ≥290 nm in the presence of 0.2% acetophenone, which sensitizes the formation of thymine-thymine but not cytosine-containing dimers; the specific mutation frequency was 7.2-fold lower compared to the frequency produced by irradiation in the absence of the photosensitizer, confirming that cytosine dimers are a major source of mutations. These results undermine the basis for the two-step model of UV mutagenesis in which a distinctly separate misincorporation step is supposed to precede the lesion bypass step; instead the results support a different two-step model, in which a deamination step precedes the bypass. The S13 capsid appears to completely inhibit the putative deamination reaction at about 75% of the dimer sites.     

Mutation probe of gene structure in E. coli: suppressor mutations in the seven-tRNA operon

Summary Cells defective in uracil-DNA glycosylase (ung:: Tn10) were used in two ways to reveal differences in select point mutations (GC to AT transitions) within the seven-tRNA operon of E. coli. The mutations were indicated as de novo or converted glutamine tRNA suppressor mutations in the genes glnU and/or glnV: (1) the kinetics of photoenzymatic monomerization of pyrimidine dimers quantitated by ung-dependent UV mutagenesis indicated more rapid repair of dimers at sites for converted suppressor mutation than of dimers at sites for de novo suppressor mutation, and (2) spontaneous deamination of cytosine was considerably more frequent at sites for converted suppressor mutation than at sites for de novo suppressor mutation. To explain these results we suggest the physical structure of the DNA in vivo is different at different sites in the seven-tRNA operon. The non-transcribed strand including specifically the anticodon region of the site for converted suppressor mutation may frequently be looped out in a single strand so that a T=C dimer is more accessible to DNA photolyase or a free cytosine residue of non-irradiated DNA is in an aqueous environment conducive to deamination. In addition, we analysed the spontaneous de novo suppressor mutation data to determine an estimate for the in vivo rate of cytosine deamination in double strand DNA of 3.2×10¹³/sec.     

Inhibition of DNA repair by trifluoperazine

We examined the possible role of calmodulin in the excision repair of ultraviolet light-induced pyrimidine dimers in damaged DNA by means of specialized assay systems. These assays included bromodeoxyuridine photolysis, dimer chromatography and cytosine arabinoside incorporation in conjunction with hydroxyurea. The calmodulin antagonist, trifluoperazine, and the calcium-chelating agent, EGTA, were employed to ascertain what affect calmodulin played in the repair process. Normal human fibroblast cells were used in all studies described in this report. After exposure to 10 J/m2 of 254 nm light, we observed a decrease of about 30% in the number of single-strand breaks produced in the presence of 25 microM trifluoperazine (1.9 vs. 3.3) in controls although the numbers of bases re-inserted in the repaired regions were similar (64 vs. 72). Measurement of thymine-containing dimers remaining throughout a 24 h time period indicated a 30% difference in the excision of dimers when tested with either EGTA or trifluoperazine. We also observed a significant decrease in the number of cytosine arabinoside arrested repair sites in the presence of either EGTA or trifluoperazine. The results are discussed with relation to the possibility of calmodulin altering the initial incision by repair endonuclease.     

Mechanism of ultraviolet-induced mutagenesis: the coding properties of ultraviolet-irradiated poly(dC) replicated by E. coli DNA polymerase I.

We have identified three lesions rather than cyclobutane dimers which alter the properties of UV-irradiated poly(dC) as a template for E.coli DNA polymerase I, and have characterised these lesions with respect to their coding properties, rates of formation and decay, and their sensitivity to uracil DNA glycosylase. Our results lead us to conclude that these lesions are (1) cytosine hydrates, which code for cytosine and to a lesser extent thymine, (2) uracil hydrates, which code for adenine and are not sensitive to uracil DNA glycosylase, and (3) uracils, which code for adenine and are removed by uracil DNA glycosylase.     

Condensation reaction of hexanucleotides containing guanine and cytosine with water soluble carbodiimide.

The condensation reactions of hexanucleotides involving guanine and cytosine in the presence of water-soluble carbodiimide (WSC) have been investigated as a model reaction of the prebiotic formation of RNA under primitive earth. The reactions formed cyclic hexanucleotides and dimers in which the product yields were dependent on the sequence.     

Potential of mean force calculations of the stacking-unstacking process in single-stranded deoxyribodinucleoside monophosphates.

The free energy of the stacking-unstacking process of deoxyribodinucleoside monophosphates in aqueous solution has been investigated by potential of mean force calculations along a reaction coordinate, defined by the distance between the glycosidic nitrogen atoms of the bases. The stacking-unstacking process of a ribodinucleoside monophosphate was observed to be well characterized by this coordinate, which has the advantage that it allows for a dynamical backbone and flexible bases. All 16 naturally occurring DNA dimers composed of the adenine, cytosine, guanine, or thymine bases in both the 5' and the 3' positions were studied. From the free-energy profiles we observed the deepest minima for the stacked states of the purine-purine dimers, but good stacking was also observed for the purine-pyrimidine and pyrimidine-purine dimers. Substantial stacking ability was found for the dimers composed of a thymine base and a purine base and also for the deoxythymidylyl-3',5'-deoxythymidine dimer. Very poor stacking was observed for the dCpdC dimer. Conformational properties and solvent accessibility are discussed for the stacked and unstacked dimers. The potential of mean force profiles of the stacking-unstacking process for the DNA dimers are compared with the RNA dimers.     

Thermal resistance to photoreactivation of specific mutations potentiated in E. coli B/r ung by ultraviolet light

Summary Mutagenesis by ultraviolet light was studied in a strain of E. coli ung, which lacks uracil-DNA glycosylase activity. Mutation potentiated by UV in cells already induced by nalidixic acid treatment was still photoreversible suggesting that pyrimidine dimers act directly as premutational photoproducts. Secondly, irradiated cells were held in buffer at 48°C for 0 to 135 min to allow for deamination of cytosines in pyrimidine dimers. The mutation frequencies for class 2 de novo suppressor mutation, for class 2 converted suppressor mutation and for backmutation were individually determined, before and after photoreactivation, as a function of this thermal treatment. Backmutation remained sensitive to photoreactivation throughout the treatment but de novo and converted suppressor mutations rapidly developed resistance to photoreactivation. This resistance was not seen in an ung ⁺ control. A model is proposed to account for the selective resistance based on the hypothesis that class 2 de novo and converted suppressor mutations normally result from UV by GC to AT transitions at T=C dimers. The model describes deamination of the cytosine residues in these dimers to become uracil residues. In consequence, monomerization by photoreactivation in cells that can not repair uracils in DNA no longer reverses mutation and GC to AT transitions are established at the sites of uracils.     

Binding of DNA to alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species prevents formation of cytosine dimers, cytosine-thymine dimers, and bipyrimidine photoadducts after UV irradiation.

Small, acid-soluble proteins (SASP) of the alpha/beta-type from spores of Bacillus and Clostridium species bind to DNA; this binding prevents formation of cyclobutane-type thymine dimers upon UV irradiation, but promotes formation of the spore photoproduct, an adduct between adjacent thymine residues. alpha/beta-Type SASP also bound to poly(dG).poly(dC) and poly(dA-dG).poly(dC-dT). While UV irradiation of poly(dG).poly(dC) produced cyclobutane-type cytosine dimers as well as fluorescent bipyrimidine adducts, the yields of both types of photoproduct were greatly reduced upon irradiation of alpha/beta-type SASP-poly(dG).poly(dC) complexes. UV irradiation of poly(dA-dG).poly(dC-dT) produced a significant amount of a cyclobutane dimer between cytosine and thymine, as well as a 6-4 bipyrimidine adduct. Again, binding of alpha/beta-type SASP to poly(dA-dG).poly(dC-dT) greatly reduced formation of these two photoproducts, although formation of the cytosine-thymine analog of the spore photoproduct was not observed. These data provide further evidence for the dramatic change in DNA structure and photoreactivity which takes place on binding of alpha/beta-type SASP and suggest that binding of these proteins to DNA in vivo prevents formation of most deleterious photoproducts upon UV irradiation.     

Short unligated sticky ends enable the observation of circularised DNA by atomic force and electron microscopies.

A comparative study of the stabilisation of DNA sticky ends by divalent cations was carried out by atomic force microscopy (AFM), electron microscopy and agarose gel electrophoresis. At room temperature, molecules bearing such extremities are immediately oligomerised or circularised by addition of Mg2+or Ca2+. This phenomenon, more clearly detected by AFM, requires the presence of uranyl salt, which stabilises the structures induced by Mg2+or Ca2+. DNA fragments were obtained by restriction enzymes producing sticky ends of 2 or 4 nucleotides (nt) in length with different guanine plus cytosine (GC) contents. The stability of the pairing is high when ends of 4 nt display a 100% GC-content. In that case, 95% of DNA fragments are maintained circular by the divalent cations, although 2 nt GC-sticky ends are sufficient for a stable pairing. DNA fragments with one blunt end and the other sticky appear as dimers in the presence of Mg2+. Dimerisation was analysed by varying the lengths and concentrations of DNA fragments, the base composition of the sticky ends, and also the temperature. Our observation provides a new powerful tool for construction of inverted dimers, and circularisation, ligation analysis or short bases sequence interaction studies.     

Evidence for a near UV-induced photoproduct of 5-hydroxymethylcytosine in bacteriophage T4 that can be recognized by endonuclease V

Summary Non-photoreactivable endonuclease V-sensitive sites have been detected in the DNA of wild type bacteriophage T4 irradiated with near UV light (320 nm). Such sites were not detected in the DNA of (a) wild type T4 irradiated with far UV (254 nm) or (b) in T4 mutants in which non-glucosylated 5-hydroxy-methylcytosine (5HMC) or cytosine replaces glucosylated 5HMC normally present in T4, irradiated with 320 nm or 254 nm light. Although the non-photoreactivable sites accounted for 50% of the endonuclease V-sensitive sites in the DNA of glucosylated T4 irradiated with near UV, there was very little difference in the sensitivities of T4 containing glucosylated 5HMC, non-glucosylated 5HMC and cytosine to near UV (313 nm). We propose that the photoproduct responsible for the non-photoreactivable, but endonuclease V-sensitive, sites in glucosylated DNA is formed from glucosylated 5HMC and that a similar photoproduct is formed from non-glucosylated 5HMC or cytosine in the appropriate phage strains. We further propose that the glucosylated 5HMC photoproduct is non-photoreactivable whereas the cytosine and non-glucosylated 5HMC photoproducts are photoreactivable and are therefore possibly cyclobutane dimers.AECL Refence No. 6370Communicated by B.A. Bridges     

Benzyl chloride and 4-chloromethylbiphenyl induced DNA damage and its repair in excision-deficient (XP group A) or -proficient human cells

Benzyl chloride (BC) and 4-chloromethylbiphenyl (4CMB) induce a class of alkaline-stable DNA damage in human cells which, like UV-induced pyrimidine dimers, undergoes repair at a slow rate by an excision-repair pathway which can be inhibited by cytosine arabinoside (araC). In the present study, in an attempt to clarify whether BC and 4CMB are UV-like agents, the excision-deficient xeroderma pigmentosum complementation group A fibroblasts and excision-proficient human alveolar tumour cells (A549) were exposed to various doses of these compounds prior to monitoring the inhibition of cell growth, DNA damage and DNA repair. The data indicate that such XP fibroblasts repair BC- and 4CMB-induced DNA damage at a normal rate, which suggests that the alkaline-stable DNA adducts induced by these chloromethyl compounds and the UV-induced pyrimidine dimers are processed by distinct excision-repair mechanisms in human cells.     

Excision repair of UV-damaged plasmid DNA in Xenopus oocytes is mediated by DNA polymerase alpha (and/or delta).

We studied DNA repair by injecting plasmids containing random pyrimidine dimers into Xenopus oocytes. We demonstrated excision repair by recovering plasmids and analyzing them with T4 UV endonuclease treatment and alkaline agarose gel electrophoresis. The mechanism for excision repair of these plasmids appears to be processive, rather than distributive, since repair occurs in 'all or none' fashion. At less than 4-5 dimers/plasmid, nearly all repair occurs within 4-6 hours (approximately 10(10) dimers repaired per oocyte); the oocyte, therefore, has abundant repair activity. Specific antibodies and inhibitors were used to determine enzymes involved in repair. We conclude that DNA polymerase alpha (and/or delta) is required because repair is inhibited by antibodies to human DNA polymerase alpha, as well as by aphidicolin, an inhibitor of polymerases alpha (and/or delta). Repair was not inhibited by hydroxyurea, cytosine beta-D-arabinofuranoside, or inhibitors of topoisomerase II (novobiocin). Oocyte repair does not activate semi-conservative DNA replication, nor is protein synthesis required. Photoreactivation cannot account for repair because dimer removal is independent of exogenous light.     

Effect of intramolecular interaction on the electron excitation state of nucleic acid components

A theoretical and experimental investigation of absorption and luminescence features of crystals and aggregates of nucleic acid bases were carried out. The long wavelength low intensity bands in UV-absorption nd excitation spectra, bathochromic shift of fluorescence spectra, the change of correlation between the intensity of fluorescence and phosphorescence spectra were obtained. The interpretation of these experimental results was proposed on the basis of pair interaction calculations (exciton-resonance and charge-resonance) in different conformations of cytosine dimers. The energy transfer after excitation at lambda 280 and 312 nm was investigated in nucleic acid base aggregates.     

Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain

We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers. While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site. Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis. Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)