Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [3H]Thd, [14C]orotate or with inorganic 32Pi. Expression of incorporated radioactivity as log10% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [3H]Thd was asymmetrically distributed among the isostichs, i.e., 3H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., T/C ratios for the first 10 isostichs averaged 1.43 +/- 0.08 and 1.28 +/- 0.05, depending on the method of analysis; furthermore, the T/C ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [3H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [3H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecular weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [14C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [3H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of 14C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from 32Pi, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since 32P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of 32P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the 'replitase' concept for channeling dNTPs to replicating forks.     

Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [³H]Thd, [¹⁴C]orotate or with inorganic ³²P_i. Expression of incorporated radioactivity as log₁₀% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [³H]Thd was asymmetrically distributed among the isostichs, i.e., ³H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., ratios for the first 10 isostichs averaged 1.43 ± 0.08 and 1.28 ± 0.05, depending on the method of analysis; furthermore, the ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [³H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [³H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecuar weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [¹⁴C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [³H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of ¹⁴C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from ³²P_i, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since ³²P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of ³²P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the ‘replitase’ concept for channeling dNTPs to replicating forks.     

Distribution patterns for 5-methylcytosine among apurinic DNAs from several sources

Purified DNA from the liver of rats, mice, rabbits, and guinea pigs, from guinea pig lymph nodes, from hyperplastic nodules induced in rat liver by feeding with 2-(acetylamino)fluorene, and from Escherichia coli cells was made apurinic by reaction with diphenylamine. After chromatographic separation of pyrimidine tracts (isostichs or isoplyths) according to the number of contiguous pyrimidines, semilog plots of tract frequency vs. the number of contiguous pyrimidines were linear, plots for DNA from several sources differed from one another, and all deviated significantly from randomness. Similar semilog plots for coding sequences among 60 mammalian genomes or 28 rat tissue genomes were intermediate among slopes for isolated DNA. Individual isostichs were hydrolyzed, and their constituent pyrimidine bases were analyzed by high-pressure liquid chromatography. Among isostichs from isolated DNAs, the distribution of Thy and Cyt contents differed markedly from the distribution of 5-methylcytosine (5-Me-Cyt); e.g., although isostich 1 contained 45-49% of 5-Me-Cyt, amounts of Thy or Cyt did not exceed 25%. Semilog plots of normalized values for tract frequency or the content of 5-Me-Cyt vs. isostich number were essentially superimposable; thus, among the first five pyrimidine tracts of a particular tissue or E. coli DNA, the number of tracts per 5-Me-Cyt moiety was essentially constant. The data showed that 5-Me-Cyt and/or dCyd-dGuo dinucleotides have a distribution throughout DNA structure that superimposes the distribution of pyrimidine tract frequency and suggests that regulatory 5-Me-Cyt moieties are principally located at 3' termini of pyrimidine tracts.     

The interpretation of oligonucleotide maps. A theoretical study of nucleic acid digests with special reference to repeated diverged sequences

Oligonucleotide mixtures produced by the digestion of RNA by specific nucleases can be defined in terms of isostichs (sets of common chain length), compositional isomers, and sequence isomers. Equations are derived to express the distribution of radioactivity on the isostich length, the distribution of compositional isomers on the isostich length, and the distribution of compositional isomers on the proportion of total radioactivity (“intensity”). It is shown how the properties of a “fingerprint” may be calculated from first principles, and conversely how the complexity of a sequence may be estimated from its fingerprint. The equations are tested by means of computer simulations of RNA digestions and their range of applicability is determined. The three distributions are used to analyze the digests of repetitious sequences. It is shown how the parameters of a diverged sequence family can, in a favorable case, be deduced from its fingerprint.     

Changes in the methylated sequences and molecular population of wheat DNA during germination

The fractions of unique (Cot less than 405), moderately (Cot=0.13--405) and highly reiterated (Cot less than 0--0.13) sequences were isolated from DNA of wheat seeds and 3 day old seedlings, and GC content, amount of 5-methylcytosine and its distribution among various pyrimidine isostichs in the fractions isolated were studied. Different in Cot value DNA fractions from seeds or from seedlings are similar in GC content and in all other characteristics studied. Seed DNA differs from DNA of seedlings in the content of pyrimidine isostichs from the respective fractions of reiterated sequences. Pronounced differences in the amount of pyridmidine clusters with various base composition in the corresponding fractions of DNA from seeds and seedlings were found. These differences in the frequencies of respective pyrimidine clusters from DNA of seeds and seedlings may be considered as being a result of changes in the molecular population of wheat DNA on germination. The seed and seedling DNA differ significantly in the 5-methylcytosine content in the respective pyrimidine isostichs isolated from unique sequences. In the seedling DNA some other nucleotide sequences are to be methylated as compared to DNA of dormat seeds. Thus, on germination some changes occur in DNA methylation as well as in the genome organization.     

Preparative isolation of di-, tri- and tetrapyrimidine nucleotides from depurinated herring sperm DNA (author's transl)

The pyrimidine nucleotides p(dT)3, p(dT)2p, pdCp, p(dC)2p and p(dC)3 and the mixtures of sequence isomers p(dC, dT), p(dC, dT)p, p(dT2, dU), (dT2, dU)p, p(dC, dT2), p(dC, dT2)p, p(dC, dT3), p(dC, dT), p(dC2, dT)p, and p(dC2, dT2) are isolated on a preparative scale from depurinated hydrolysates of herring sperm DNA by the following procedure. The DNA hydrolysate is first separated into a low and a high molecular weight mixture of pyrimidine nucleotides by column chromatography on DEAE-cellulose. The nucleoside bisphosphates pdCp and pdTp, the dinucleotides p(dC)2 and p(dT)2 and the sequence isomers p(dC, dT) are largely separated out of the mixture of the low molecular weight pyrimidine nucleotides. The remaining mixture is rechromatographed at pH 3.5 on QAE-Sephadex. This separates the pyrimidine nucleotides containing a majority of cytidylic acid units from those containing more thymidylic acid units, which are then fractionated at pH 7.5 according to the number of bases in the chain. The pyrimidine nucleotides and mixtures of sequence isomers separated to 83-99% purity by column chromatography are further separated by paper chromatography and are obtained in chromatographically pure form after this step. The structures of the isolated DNA fragments are determined from chromatographic data, absorption and enzymatic degradation.     

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.     

DNA-methylase from regenerating rat liver: purification and characterisation.

DNA methylase has been purified 660-fold from nuclei from regenerating rat liver. The enzyme is able to methylate single stranded (ss) and double stranded (ds) DNA, the only reaction product being 5-methylcytosine. Previously unmethylated double stranded DNA from prokaryotes (M.luteus) as well as from eukaryotes (Ascaris suis) can serve as substrates. The synthetic copolymers (dG-dC)n . (dC-dG)n and (dG,dC)n are also methylated. While SV40 DNA is almost not methylated, PM2 DNA is a good substrate even in the supercoiled form. The enzyme methylates 1 in 17 bases in heterologous M.luteus DNA, but only 1 in 590 in homologous rat liver DNA. The high methylation level of M.luteus DNA, an analysis of the methylated pyrimidine isostichs and a preliminary dinucleotide analysis suggest that all the CpGs in a DNA can be methylated.     

Distribution of pyrimidine sequences of different length and base composition in bacteriophage T5 DNA]

Distribution of pyrimidine tracts of different length (isopliths) with general formula PynPn+1 in bacteriophage T5 DNA was studied. The first seven isoplith fractions were subfractionated by the chain length and the quantity of the resulting non-isomeric oligonucleotides was determined. The pattern of distribution of pyrimidine tracts of various length and base composition in bacteriophage T5 DNA is different from that previously observed in the DNAs of bacteriophages T3 and T7. The observed differences in distribution of pyrimidine nucleotides are in accordance with the other peculiarities of bacteriophage T5 genome.     

DNA-methylase activities from animal mitochondria and nuclei: different specificity of DNA methylation]

DNA-methylase activities which methylate cytosine residues in homo- and heterologous DNA were detected in mitochondria and nuclei from rat liver and beef heart. Adenine modifying DNA-methylases in mitochondria and nuclei were not found. DNA from mitochondria and nuclei differ significantly in the methylation degree and in the pattern of the 5-methyl-cytosine distribution by pyrimidine isostichs as DNA in vivo and in vitro being methylated. Mitochondrial DNA methylase has the maximum activity at 30 degrees and pH 7.8 this enzyme(s) differ(s) from the nuclear one(s) in the pH dependence of its activity. After exhaustive in vitro methylation of various DNA by the nuclear enzyme DNA-methylase from mitochondria additionally introduces CH3 groups from S-adenosylmethionine into these DNA (about 3 times more CH3 groups than nuclear enzyme). Nuclear DNA-methylase also methylates DNA which is previously fully-methylated by the mitochondrial enzyme, but to a lesser degree. In conditions of exhaustive DNA methylation mitochondrial enzyme introduces into E. coli B DNA about four times more methyl groups as compared to the nuclear one. After the methylation of E. coli B DNA by mitochondrial enzyme the label (3H-methyl) was detected predominantly in mono-, and in case of nuclear enzyme--in di- and tripyrimidine fragments. Mitochondrial DNA-methylase differs from the nuclear one in the nature of recognized DNA sequences; these enzymes seems to be represented by different proteins. The mitochondrial enzyme methylates shorter nucleotide sequences in DNA as compared to the nuclear DNA-methylase. All these data suggest there exist organoid specificity of genome methylation in animal cell and the modification-restriction systems in animal nucleus and mitochondria are different in character.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.     

Formation of the main UV-induced thymine dimeric lesions within isolated and cellular DNA as measured by high performance liquid chromatography-tandem mass spectrometry

UVB radiation-induced formation of dimeric photoproducts at bipyrimidine sites within DNA has been unambiguously associated with the lethal and mutagenic properties of sunlight. The main lesions include the cyclobutane pyrimidine dimers and the pyrimidine (6-4) pyrimidone adducts. The latter compounds have been shown in model systems to be converted into their Dewar valence isomers upon exposure to UVB light. A new direct assay, based on the use of liquid chromatography coupled to tandem mass spectrometry, is now available to simultaneously detect each of the thymine photoproducts. It was applied to the determination of the yields of formation of the thymine lesions within both isolated and cellular DNA exposed to either UVC or UVB radiation. The cis-syn cyclobutane thymine dimer was found to be the major photoproduct within cellular DNA, whereas the related (6-4) adduct was produced in an approximately 8-fold lower yield. Interestingly, the corresponding Dewar valence isomer could not be detected upon exposure of human cells to biologically relevant doses of UVB radiation.     

Activity assay of His-tagged E. coli DNA photolyase by RP-HPLC and SE-HPLC

Escherichia coli DNA photolyase was expressed as C-terminal 6x histidine-fused protein. Purification of His-tagged E. coli DNA photolyase was developed using immobilized metal affinity chromatography with Chelating Sepharose Fast Flow. By one-step affinity chromatography, approximate 4.6 mg DNA photolyase was obtained from 400 ml E. coli culture. The purified His-tagged enzyme was combined with two chromophors, FADH and MTHF. Using the oligonucleotide containing cyclobutane pyrimidine dimer as substrate, both reversed-phase high-performance liquid chromatography and size-exclusion high-performance liquid chromatography were developed to measure the enzyme activity. The enzyme was found to be able to repair the cyclobutane pyrimidine dimer with the turnover rate of 2.4 dimers/photolyase molecule/min.     

Turnover of carnitine by rat tissues.

A small release of Pi from a diphenylamine-formic acid digest of DNA was detected after elimination of interpurine phosphodiester bonds was complete. Minor components in the DNA digest were identified as pyrimidine oligonucleotides which had lost one terminal phosphate. Isolated pyrimidine tracts released Pi on redigestion with the formic acid-diphenylamine reagent in amounts that increased with the number of nucleotides in the oligonucleotide taken. The oligonucleotides were also partially degraded by the formic acid-diphenylamine reagent and the degradation (2-3% of phosphodiester bonds between consecutive nucleotides) was almost independent of chain length. The cleavage was random with no preference for a phosphodiester bond flanked by particular nucleosides. This minor lack of specificity in the formic acid-diphenylamine-catalysed degradation of DNA can, however, account for the low recoveries of long pyrimidine tracts previously reported. Any analysis of pyrimidine tracts in a DNA molecule should make some correction for this small degree of degradation if exact assignments of the numbers of pyrimidine tracts are to be made.     

Synthesis and binding properties of oligo-2'-deoxyribonucleotides conjugated with triple-helix-specific intercalators: benzo[e] and benzo[g] pyridoindoles

DNA binding compounds, such as benzo[e] (BePI) and benzo[g] pyridoindole (BgPI) derivatives, exhibit preferential stabilization of triple helices. We report here the synthesis of a series of pyrimidine triple-helix-forming oligo-2'-deoxyribonucleotides conjugated with these molecules. BePI was coupled to the 5-position of 2'-deoxyuridine via two linkers of different sizes attached to its 11-position and placed at either the 5'-end, inside the sequence, or at both the 5'-end and the internal positions using periodate oxidation of a diol-containing oligonucleotide followed by reductive coupling with amino-linked BePI. The same BePI derivatives were also linked to the oligonucleotide chain via internucleotidic phosphorothiolate or phosphoramidate linkages. A mixture of diastereoisomers was prepared as well as separate pure Rp and Sp isomers. A BePI derivative, with two different linkers attached to its 3-position, and BgPI derivatives were also linked to the 5-position of a 2'-deoxyuridine located at either the 5'-end or inside the sequence, as well as to the beta- anomeric position of an additional 2'- deoxyribose placed inside the sequence. The binding properties of these oligonucleotide-benzopyridoindoles conjugates with their double-stranded DNA target was studied by absorption spectroscopy.     

Synthesis and Substrate Properties of Modified Nucleoside 5"-Triphosphates Mimicking dATP in the Reactions of DNA Synthesis Catalyzed by DNA Polymerases

Several modified nucleoside 5"-triphosphates containing adenine-mimicking pyrimidine derivatives as an aglycone were synthesized. The study of substrate properties of these compounds towards DNA-synthesizing enzymes showed their ability of being incorporated into the growing DNA chain in place of dATP.     

Synthesis and substrate properties of modified nucleoside 5'-triphosphates mimicking dATP in the reactions of DNA synthesis catalyzed by DNA polymerases

Several modified nucleoside 5'-triphosphates were synthesized containing adenine-mimicking pyrimidine derivatives as an aglycone. The study of substrate properties of these compounds towards DNA-synthesizing enzymes showed their ability of being incorporated into the growing DNA chain in place of dATP.     

The kinetics of fluorescent DNA labeling using PCR with different Taq polymerases depends on the chemical structures of modified nucleotides

The kinetics of DNA labeling during PCR using six fluorescent derivatives of 2′-deoxyuridine 5′-triphosphate has been studied. These compounds differ in their chemical structure, total electric charge and the length of the linker between a dye and the C5 position of a pyrimidine base. The efficiency of the incorporation of the fluorescent derivatives into a growing DNA chain by four commercially available Taq DNA polymerases with 5′→3′ exonuclease and hot start activity has been determined using real-time PCR with a TaqMan probe and the subsequent electrophoretic analysis of the reaction products. Modified deoxyuridines with a total positive or negative charge of the chromophore were practically not incorporated by Taq polymerases during PCR. The modified deoxyuridines with a neutral charge of the chromophore were effectively incorporated into DNA. The extended length of the linker between the pyrimidine base and the chromophore led to a lower PCR inhibition and a more effective inclusion of modified nucleotides in the growing DNA chain. This fact can be explained by the reduced steric effects that were caused by the dye. As a result, the most promising combinations of fluorescently labeled nucleotide and Taq polymerase have been chosen for further use in fluorescent DNA labeling.