Mechanisms of targeted frameshift mutations: Insertions arising during error-prone or SOS synthesis of DNA containing cis-syn cyclobutane thymine dimers

It is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The polymerase model is commonly used to explain the mechanisms of various mutations. An alternative polymerase-tautomer model was developed for UV-induced mutagenesis. A mechanism was proposed for targeted insertions caused by cis-syn cyclobutane thymine dimers. Targeted insertions are frameshift mutations due to addition of one or more nucleotides in a DNA sequence opposite to a lesion capable of stopping DNA synthesis. Among other factors, cyclobutane pyrimidine dimers can cause targeted insertions. UV irradiation can change the tautomeric form of DNA bases. Five rare tautomeric forms are possible for thymine, and they are stable when the thymine is a component of a cyclobutane dimer. A structural analysis showed that none of the canonical nucleotides can be added opposite to a specific rare thymine tautomer so that hydrogen bonds form between the two bases. A single nucleotide gap is consequently left in the corresponding site of the nascent strand when a specialized or modified DNA polymerase drives SOS or error-prone DNA synthesis on a template containing cis-syn cyclobutane thymine dimers with a base occurring in the rare tautomeric form. If the DNA composition is homogenous within the region, the end of the growing DNA strand may slip to form a complementary pair with the nucleotide adjacent to the dimer according to the Streisinger model, thus producing a loop. A targeted insertion is thereby generated to make the daughter strand longer. Targeted insertions were for the first time assumed to result from the cis-syn cyclobutane thymine dimers wherein one or both of the bases occur in the specific tautomeric form that does not allow the addition and hydrogen bonding of any canonical nucleotide in the opposite position. A model was developed to explain how targeted insertions of one or more nucleotides are caused by cis-syn cyclobutane thymine dimers. Thus, the polymerase-tautomer model can explain the nature and formation of targeted frameshift mutations in addition to hot and cold spots or targeted or untargeted nucleotide substitutions.     

Analysis of DNA Repair and Protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after Exposure to UVC Radiation

Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m² of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy.     

Measurement of thymine dimers in DNA by thin-layer chromatography: II. The use of one-dimensional systems

A number of chromatographic solvents and thin-layer chromatographic matrices were tested for their effectiveness in separating thymine dimers from thymine by one-dimensional chromatography. One combination, viz., watersaturated ethylacetate:n-propanol on silica gel thin layers was found to be particularly useful.     

Gas chromatographic—mass spectrometric determination of plasma 5-fluorouracil after administration of 1-hexylcarbamoyl-5-fluorouracil to dogs and humans

A simple, sensitive and specific method for determining 5-fluorouracil (5-FU) in plasma after the administration of 1-hexylcarbamoyl-5-fluorouracil (HCFU) was developed using gas chromatography—mass spectrometry. Thymine was used as the internal standard. After removal of interfering substances with chloroform, diethyl ether and Amberlite XAD-2 resin, 5-FU and thymine were extracted with 16% n-propanol in diethyl ether and methylated with trimethylanilinium hydroxide. Fragment ions at m/e 158 and 154, the molecular ion of the dimethyl derivatives of 5-FU and thymine, respectively, were used to monitor 5-FU and thymine. The sensitivity of the method is 10 ng/ml, which is sufficient to determine the 5-FU levels in plasma after the administration of therapeutic doses of HCFU to patients.     

SYNTHESIS AND ANTIPROLIFERATIVE ACTIVITY OF SOME 4′-C-HYDROXYMETHYL-α- AND -β-D-ARABINO-PENTOFURANOSYL PYRIMIDINE NUCLEOSIDES

A suitably protected 4-C-hydroxymethyl-arabino-pentofuranose was prepared and condensed with the following nucleobases: uracil, 5-fluorouracil and thymine. The corresponding cytosine and 5-fluorocytosine derivatives have also been obtained respectively from the uracil and 5-fluorouracil nucleosides. Separation of the anomeric mixtures followed by deprotection afforded the target compounds that were found to be non-cytotoxic to CCRF-CEM leukemia cells.     

Selective hydrolysis of damaged DNA by nuclease P1

The turnover rates for hydrolysis by nuclease P1 of the 16 unmodified dideoxynucleoside monophosphates were measured. In addition, the turnover rates were measured in a variety of dideoxynucleoside monophosphates containing free radical-induced base modifications. The modified bases included cis-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), 5,6-dihydrothymine, 5-hydroxymethyuracil, 8-hydroxyguanine, 5-hydroxy-5-methylhydantoin and the formamido remnant which can be derived from either a thymine or a cytosine base. The turnover rate for dinucleoside monophosphates containing 4,8-dihydro-4-hydroxy-8-oxo-guanine modifications, which are induced by singlet oxygen, were also measured. A model was devised for the hydrolysis of DNA by nuclease P1 which uses the observed turnover rates as parameters. The model predicts the abundance of monomers and dimers as hydrolysis proceeds. Whereas the level of monomers increases monotonically, the level of each dimer first increases and then falls off. There are advantages to phosphorylating dimers, as compared with monomers, using polynucleotide kinase. Consequently this model may be of interest in connection with ³²P-postlabeling applied to the measurement of DNA damage in nuclease P1 partial hydrolysates of DNA.     

The biosynthesis of natural and unnatural polyoxins by Streptomyces cacaoi

The biosynthesis of the pyrimidine moiety and the uronic acid moiety of the polyoxins and the formation of unnatural polyoxins has been studied in Streptomyces cacaoi. Experimental evidence is provided for the biosynthesis of thymine via a pathway that is independent of thymidylate synthetase. This new thymine pathway is based on two experimental approaches. First, two known inhibitors of DNA synthesis (1-formylisoquinoline thiosemicarbazide and 5-fluoro-2′-deoxyuridine), when added to polyoxin-producing cultures of S. cacaoi, inhibit the synthesis of TMP from exogenously supplied uracil but do not inhibit the synthesis of the thymine or hydroxymethyluracil in the polyoxin complex. Second, exogenously supplied thymine and hydroxymethyluracil are taken up by S. cacaoi but are not incorporated into the thymine or hydroxymethyluracil of the polyoxin complex. The thymine is incorporated into the DNA. The uracil in polyoxin L could be the parent pyrimidine chromophore with C-1 additions occurring at carbon-5 to form thymine and hydroxymethyluracil. Carbon-3 of serine but not the methyl group of methionine is a one-carbon source for the formation of the thymine and hydroxymethyluracil in the polyoxin complex.S. cacaoi can synthesize unnatural polyoxins, as evidenced by the incorporation of 5-fluoro, 5-bromo, and 6-azauracil into the polyoxins; 5-iodo-, 2-thio-, or 4-thiouracil is not a substrate. Two new polyoxin analogs synthesized and characterized when 5-fluorouracil is added to the cultures are 5-fluoropolyoxin L and 5-fluoropolyoxin M. There is a marked change in the molar ratio of the uracil:thymine:hydroxymethyluracil chromophores in the polyoxin complex following the incorporation of 5-fluoro-, 5-bromo-, or 6-azauracil. Apparently, the unnatural polyoxins inhibit the addition of the C-1 unit to carbon-5 of uracil in the polyoxin complex. Polyoxin L and polyoxin C do not inhibit Escherichia coli and Streptococcus faecalis, but 5-fluoropolyoxin L and 5-fluoropolyoxin C inhibit both these organisms. There is little or no difference in the inhibition of the fluorinated and natural polyoxins against leukemia L-1210 cells. The fluoro group on carbon-5 of the uracil ring does not affect the enzyme-inhibition complex with chitin synthetase since the inhibition constant of fluoropolyoxins L is the same as has been reported for polyoxins A, D, and L.The ¹⁴C-labeling pattern in the 5′-amino-5′-deoxy-d-allofuranosyluronic acid moiety of the polyoxins from ¹⁴C-labeled glucose, allose, and glycerol suggests that the formation of this unique C-6 uronic acid in the polyoxins does not proceed via the direct oxidation of either d-glucose or d-allose to the -onic or -uronic acids. Glucose is converted to two three-carbon trioses, followed by either (i) the oxidation of one of the trioses to a threecarbon acid and subsequent condensation with another three-carbon sugar to form the C-6 uronic or (ii) an 80:20 equilibrium of the two trioses followed by condensation to a hexose which is then oxidized to the C-6 uronic acid.     

Analysis of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogen during the cell cycle. II. Ultraviolet light

UV-induction of thymine dimers in cellular DNA and their excision during different phases of the cell cycle of HeLa S3 cells were studied. Induction of thymine dimers was higher in the mitotic phase and the middle of the S phase than in the G₁ phase and from the late S phase to the early G₂ phase which are rather insensitive to UV. However, there is no significant difference in excision rate of UV-induced thymine dimers from the irradiated cells through the cell cycle. These findings indicate that the cyclic variation of UV-survivals during the cell cycle may be due to differences in the amount of thymine dimers in cellular DNA induced by UV-irradiation.     

The Ultraviolet Photochemistry and Photobiology of Vegetative Cells and Spores of Bacillus megaterium

The ultraviolet (UV) photochemistry and photobiology of spores and vegetative cells of Bacillus megaterium have been studied. The response of vegetative cells of B. megaterium appears qualitatively similar to those of Escherichia coli, Micrococcus radiodurans, and Bacillus subtilis with respect to photoproduct formation and repair mechanisms. UV irradiation, however, does not produce cyclobutane-type thymine dimers in the DNA of spores, although other thymine photo-products are produced. The photoproducts do not disappear after photoreactivation, but they are eliminated from the DNA by a dark-repair mechanism different from that found for dimers in vegetative cells. Irradiations performed at three wavelengths produce the same amounts of spore photoproduct and give the same survival curves. Variation of the sporulation medium before irradiation results in comparable alterations in the rate of spore photoproduct production and in survival.     

Excision Repair Properties of Isogenic rec− Mutants of Escherichia coli K-12

We have examined the excision repair properties of isogenic rec⁻ and uvr⁻ strains of Escherichia coli K-12. A recB⁻recC⁻ strain excises dimers at a rate nearly that of the rec⁺ parent, reaching the same extent of excision after a 1-hr postirradiation incubation. recA⁻ and recA⁻recB⁻ strains excise 75 to 80% of the dimers excised by their rec⁺ parent, whereas a uvrB⁻ strain excises no dimers during a 1-hr incubation. The doses of ultraviolet light (254 nm) required to reduce survival to 37% of the original population are 8 ergs/mm² for recA or recA recB mutants, 5 ergs/mm² for the uvrB⁻ strain, 30 ergs/mm² for the recB recC mutant, and 230 ergs/mm² for the wild-type parent. From these data one cannot account for the ultraviolet light sensitivity of rec⁻ strains on the basis of their excision repair properties. We conclude that rec gene products play no significant role in the early steps of excision repair. The assay we have used for excision of thymine dimers is a modification of the Carrier-Setlow technique, and is described in detail in the Appendix to this paper. To show the properties and validity of this method, results of experiments with thymine dimers formed in vitro and in vivo in E. coli K-12 are presented. These results show our method to be reproducible and sensitive to 0.005% of the total radioactive thymine present in thymine-containing dimers.     

Predicting thymine dimerization yields from molecular dynamics simulations

It was recently shown that thymine dimers in the all-thymine oligonucleotide (dT)₁₈ are fully formed in <1 ps after ultraviolet excitation. The speed and low quantum yield of this reaction suggest that only a small fraction of the conformers of this structurally disordered oligonucleotide are in a position to react at the instant of photon absorption. In this work, we explore the hypothesis that conventional molecular dynamics simulations can be used to predict the yield of cyclobutane pyrimidine dimers in DNA. Conformations obtained from simulations of thymidylyl-(3′-5′)-thymidine in various cosolvents were classified as dimerizable or nondimerizable depending on the distance between the C5-C6 double bonds of the adjacent thymine bases and the torsion angle between them. The quantum yield of cyclobutane pyrimidine dimer formation was calculated as the number of dimerizable conformations divided by the total number of conformations. The experimental quantum yields measured in the different solvents were satisfactorily reproduced using physically reasonable values for the two parameters. The mean dimerizable structure computed by averaging all of the dimerizable cis-syn conformations is structurally similar to the actual cis-syn dimer. Compared to the canonical B-form TT step, the most important structural property of a dimerizable conformation is its reduced helical twist angle of 22°.     

The kinetics of thymine dimer excision in ultraviolet-irradiated human cells

We have investigated the kinetics of the loss of thymine dimers from the acid-insoluble fraction of several ultraviolet (UV)-irradiated cultured human cell lines. Our results show that UV fluences between 10 and 40 J/m2 produce an average of 21-85 x 10(5) thymine dimers per cell and an eventual maximal loss per cell of 12-20 x 10(5) thymine dimers. The time for half-maximal loss of dimers ranged from 12-22 h after UV irradiation. In contrast, the time for half-maximal repair synthesis of DNA measured by autoradiography was 4.5 h. This figure agrees well with reported half-maximal repair synthesis times, which range from 0.5 to 3.6 h based on our analysis. The discrepancy in the kinetics of the loss of thymine dimers from DNA and repair synthesis is discussed in terms of possible molecular mechanisms of thymine dimer excision in vivo and in terms of possible experimental artifacts.     

Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by $\text{E. coli}$ DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I $\text{amber}$ mutants.     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

γ-Irradiation of Thymine Dimers in Aqueous Solution

Thymine dimers were irradiated in aqueous solution with ⁶⁰Co γ-rays in N₂ or O₂. Thymine and unidentified non-UV-absorbing products appeared. The thymine was identified by spectrophotometry, chromatography, and ability to support the growth of Escherichia coli 15 T^-. Residual dimer was determined by a UV-reversibility assay. The G-values for dimer breakage were approximately equal in N₂ and O₂. At low γ-doses, about two thymines were produced per dimer broken in N₂, whereas only about one thymine appeared per dimer broken in O₂. For dimer irradiated in frozen solution, the yield of thymine was at least 100 times less than in liquid.     

Effects of temperature on the photoreactivation of ultraviolet-b–induced dna damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6-4 photoproducts) induced by ultraviolet-B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet-A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6-4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6-4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6-4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6-4 photoproducts. Significant repair of both thymine dimers and 6-4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6-4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.     

Photoreactivation in Airborne Mycobacterium parafortuitum

Photoreactivation was observed in airborne Mycobacterium parafortuitum exposed concurrently to UV radiation (254 nm) and visible light. Photoreactivation rates of airborne cells increased with increasing relative humidity (RH) and decreased with increasing UV dose. Under a constant UV dose with visible light absent, the UV inactivation rate of airborne M. parafortuitum cells decreased by a factor of 4 as RH increased from 40 to 95%; however, under identical conditions with visible light present, the UV inactivation rate of airborne cells decreased only by a factor of 2. When irradiated in the absence of visible light, cellular cyclobutane thymine dimer content of UV-irradiated airborne M. parafortuitum and Serratia marcescens increased in response to RH increases. Results suggest that, unlike in waterborne bacteria, cyclobutane thymine dimers are not the most significant form of UV-induced DNA damage incurred by airborne bacteria and that the distribution of DNA photoproducts incorporated into UV-irradiated airborne cells is a function of RH.     

Theory of co-operative formation of defects in DNA molecule under ultraviolet irradiation

The process of thymine dimers formation in DNA under the action of ultraviolet irradiation has been analyzed theoretically taking into account the long range transfer of electronic excitation along the molecule. First, for the case of homopolymer (poly dA.dT) an exact analytical solution of the problem has been obtained. For the case of random walks of excitation as well as for the case of its directional motion the distribution functions of undamaged regions on their lengths have been calculated. These functions turned out to differ drastically from a trivial exponential distribution taking place without excitation's transfer. At the same time the transfer of excitation leads to the effect of thymine dimers clustering—the new dimers are formed mainly in the vicinity of dimers formed earlier. The degree of clustering γ (the mean number of dimers per cluster) depends quadratically on the concentration of clusters C in the homopolymer for the case of random walks of excitation. For the case of a heteropolymer having inhomogeneous distribution of potential dimers (two thymines situated one by one in the same strand) a machine algorithm for numerical solution of the problem by the Monte-Carlo method has been proposed. Calculations for the heteropolymer with a random sequence of bases have shown that in this case the curve γ(c) is S-shaped and its form agrees with the experimental one obtained by Shafranovskaya, Trifonov, Lazurkin &; Frank-Kamenetskii (1972). The values of parameters providing the best agreement between theory and experiment have been obtained. Using these values of parameters the calculations of distribution function of undamaged regions and curves γ(c) for different concentrations of AT-pairs in DNA have been performed. These calculations show that the degree of clustering γ rises dramatically with increasing concentration of AT-pairs in DNA. Theoretical predictions which can be tested experimentally have been summarized.     

Studies of the induction of mitotic gene conversion by ultraviolet irradiation: II. Action spectra

Action spectra for the induction of intragenic mitotic recombination (gene conversion) at the trp5 locus by UV are presented for three cell stages (T₀, T₉ and T₁₆) taken from synchronously growing cultures of Saccharomyces cerevisiae. The spectra over the range from 230 to 300 nm were taken mostly in 5-nm steps. The peak of action spectra was significantly shifted, regardless of the stage, toward the longer wavelengths as compared with that of the absorption spectrum of DNA (258 nm) or even that of thymine (265 nm). In one extreme case (T₁₆), the peak was shifted 17 nm from the absorption peak of DNA. Further, the spectrum changed its shape at the cell stage advanced from non-dividing (unbudded) (T₀) to a dividing phase (T₁₆). Furthermore, the induction cross section decreased by a large factor (about 40), regardless of the wavelength, in going from T₀ to T₁₆. From observations of the high photoreversibility of induced conversions, the major primary damage was thought to be pyrimidine dimers in the DNA.One plausible explanation, though not quite satisfactory from the quantitative viewpoint for these findings was that the increasing RNA during growth would screen the incident UV differentially with respect to the stage. If this explanation is correct, thymine dimers may still be considered, in spite of the shifts and deformations in the action spectra, as the major primary damage that triggers the long series of processes leading to gene conversion. Conventional methods for obtaining action spectra are discussed in comparison with the present method, which was based on sensitivity parameter a in the proposed dose (t)-frequency (f) relation, f = (at)^α (α is the multiplicity parameter).     

The versatile thymine DNA-glycosylase: a comparative characterization of the human,Drosophila and fission yeast orthologs

Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G·T and G·U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species-specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4- ethenocytosine opposite guanine and 5-fluorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5′-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual species.