Metabolism of thymine nucleotides synthesized via the ''de novo'' mechanism in normal, megaloblastic and methotrexate-treated human cells and in a lymphoblastoid cell line.

Human bone-marrow cells and lymphocytes were incubated with [3H]deoxyuridine (dU) to study the metabolism of thymine nucleotides labelled via the thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) step of the 'de novo' biosynthetic pathway. (1) Continuous labelling with [3H]dU was used to compare incorporation of label into DNA with the specific radioactivities of thymine nucleotides separated by paper chromatography. (2) Cells were also labelled with [3H]dU at 13 degrees C, and 'chased' in unlabelled medium at 37 degrees C in order to quantify the proportion of thymine nucleotides incorporated into DNA and the proportion degraded. Only 40% of labelled thymine nucleotides were incorporated into lymphocyte DNA during a 'chase', whereas 100% were incorporated by MOLT 4 cells (a lymphoblastoid cell line of thymic origin, Thy-ALL line). Unincorporated nucleotides were rapidly degraded in lymphocytes, but degradative activity was very low in MOLT 4 cells. The results described here reinforce our previous conclusions [Taheri, Wickremasinghe & Hoffbrand (1981) Biochem. J. 194, 451-461] that there is a single thymine nucleotide compartment in Thy-ALL cells, but at least two pools in lymphocytes and bone-marrow cells. This compartmentation of nucleotides in human cells is consistent with a model which proposes that deoxyribonucleotides are localized near replication forks by the activity of multienzyme complexes [Mathews, North & Reddy (1978) Adv. Enz. Regul. 17, 133-156]. Our results also suggest that thymine nucleotides derived by the 'de novo' mechanism may be more highly localized than those derived by salvage. In cells from patients with megaloblastic anaemia owing to deficiency of vitamin B12 or folate or in normal cells treated with methotrexate, there was a massive accumulation of labelled dUMP and decreased incorporation of label into DNA. There was no measurable incorporation of labelled deoxyuridine residues into DNA of megaloblastic cells, but deoxyuridine residues were detected in DNA of cells treated with methotrexate.     

Monovalent cation binding by curved DNA molecules containing variable numbers of a-tracts

Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two curved ∼200 basepair (bp) restriction fragments, one taken from the M13 origin of replication and the other from the VP1 gene of SV40. These two fragments have previously been shown to contain stable, centrally located bends of 44° and 46°, respectively, located within ∼60 bp “curvature modules” containing four or five irregularly spaced A-tracts. Transient electric birefringence measurements of these two fragments, sequence variants containing reduced numbers of A-tracts in the SV40 curvature module or changes in the residues flanking the A-tracts in the M13 curvature module, have been combined with the free solution electrophoretic mobilities of the same fragments using known equations to estimate the effective charge of each fragment. The effective charge is reduced, on average, by one-third charge for each A-tract in the curvature module, suggesting that each A-tract binds a monovalent cation approximately one-third of the time. Monovalent cation binding to two or more A-tracts is required to observe significant curvature of the DNA helix axis.     

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5'exonuclease proof-reading activity of the DNA polymerase.     

Synthesis of thymine and alpha-putrescinylthymine in bacteriophage phi W-14-infected Pseudomonas acidovorans.

Host DNA synthesis stopped about 10 min after the infection of Pseudomonas acidovorans with bacteriophage phi W-14, but host DNA was not degraded to acid-soluble fragments. The synthesis of host but not of phage DNA was inhibited by 5-fluorodeoxyuridine. The nucleotide pools of infected cells did not contain dTTP, and infection resulted in the appearance of dTTPase activity. Although ornithine labeled the alpha-putrescinylthymine residues of phi W-14 DNA, ornithine-labeled nucleotides were not detected in infected cells. A new deoxynucleoside triphosphate did appear in infected cells, but it was not labeled by ornithine. It is concluded that the thymine and alpha-putrescinylthymine in phi W-14 DNA are synthesized at the polynucleotide level.     

Actions of human DNA glycosylases on uracil-containing DNA, methylated DNA and their reconstituted chromatins.

Extracts of human lymphoblastoid cells catalyzed complete release of uracil (Ura) from PBS1 DNA, which contains Ura instead of thymine as a normal component (Ura-DNA), and 3-methyladenine (3-MeAde) from DNA methylated with methyl methanesulfonate (Me-DNA). These two activities, Ura-DNA glycosylase and 3-MeAde-DNA glycosylase, differed in heat stability. Cell extracts released Ura more rapidly and 3-MeAde more slowly from alkali-denatured preparations of Ura- and Me-DNA, respectively, than from native DNA's. On incubation with reconstituted chromatins, prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein by salt gradient dialysis, cell extracts released all the Ura but only about half of the 3-MeAde residues, although both these chromatins were degraded by micrococcal nuclease until about half of the nucleotides became acid soluble. The activities of Ura-DNA and 3-MeAde-DNA glycosylase of xeroderma pigmentosum cells were similar to those of normal cells.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.     

Sequence specificity of ozone-degradation of bases in supercoiled plasmid DNA

It was found that ozone reacted preferentially with thymine and guanine residues located in the specific region in pBR322 DNA. The sequence analysis of the region including the cleavage site produced by ozonization of ccDNA showed that ozone-modification proceeded in the single stranded region formed by cruciform-formation in supercoiled DNA.     

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.     

A specific mismatch repair event protects mammalian cells from loss of 5-methylcytosine

5-Methylcytosine spontaneously deaminates to form thymine, thus generating G/T mispairs in DNA. We investigated the way in which these lesions are addressed in mammalian cells by introducing specific G/T mispairs into the genome of SV40 and determining the fate of the mismatched bases in simian cells. Mispairs were incorporated in 12 bp synthetic duplexes ligated into SV40 DNA between the BstXI and TaqI restriction sites. Analysis of 347 plaques obtained after transfection of this modified DNA indicated that mispairs were corrected in 343 cases (99%), revealing 314 repair events in favor of guanine (90%) and 29 in favor of thymine (8%). Correction in favor of guanine occurred regardless of the orientation of the mispair in DNA and regardless of whether the mispair was in the commonly methylated CpG dinucleotide. These results attest to a specific mismatch repair pathway that restores G/C pairs lost through deamination of 5-methylcytosine residues.     

Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli

The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of phi X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions. phi X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease III is involved in the repair of thymine glycols in vivo. phi X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease III and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols. phi X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease III or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other. These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV.     

Surface salt bridges modulate DNA wrapping by the type II DNA-binding protein TF1

The histone-like protein HU is involved in compaction of the bacterial genome. Up to 37 bp of DNA may be wrapped about some HU homologues in a process that has been proposed to depend on a linked disruption of surface salt bridges that liberates cationic side chains for interaction with the DNA. Despite significant sequence conservation between HU homologues, binding sites from 9 to 37 bp have been reported. TF1, an HU homologue that is encoded by Bacillus subtilis bacteriophage SPO1, has nM affinity for 37 bp preferred sites in DNA with 5-hydroxymethyluracil (hmU) in place of thymine. On the basis of electrophoretic mobility shift assays, we show that TF1-DNA complex formation is associated with a net release of only approximately 0.5 cations. The structure of TF1 suggests that Asp13 can form a dehydrated surface salt bridge with Lys23; substitution of Asp13 with Ala increases the net release of cations to approximately 1. These data are consistent with complex formation linked to disruption of surface salt bridges. Substitution of Glu90 with Ala, which would expose Lys87 predicted to contact DNA immediately distal to a proline-mediated DNA kink, causes an increase in affinity and an abrogation of the preference for hmU-containing DNA. We propose that hmU preference is due to finely tuned interactions at the sites of kinking that expose a differential flexibility of hmU- and T-containing DNA. Our data further suggest that the difference in binding site size for HU homologues is based on a differential ability to stabilize the DNA kinks.     

Biodegradation of high concentrations of formaldehyde using Escherichia coli expressing the formaldehyde dismutase gene of Methylobacterium sp. FD1

In the present study, formaldehyde dismutase from Methylobacterium sp. FD1 was partially purified and analyzed by nanoLC–MS/MS; it was then cloned from the genomic DNA of FD1 by PCR. The open reading frame of the formaldehyde dismutase gene of FD1 was estimated to be 1203 bp in length. The molecular weight and pI of formaldehyde dismutase (401 aa), as deduced from the FD1 gene, were calculated at 42,877.32 and 6.56, respectively. NAD(H)-binding residues and zinc-binding residues were found in the amino acid sequence of the deduced formaldehyde dismutase of FD1 by BLAST search. The resting Escherichia coli cells that were transformed with the FD1 formaldehyde dismutase gene degraded high concentrations of formaldehyde and produced formic acid and methanol that were molar equivalents of one-half of the degraded formaldehyde. The lyophilized cells of the recombinant E. coli also degraded high concentrations of formaldehyde.     

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.     

DNA-methyltransferase SsoII interaction with own promoter region binding site.

The investigation of Sso II DNA-methyltransferase (M.Sso II) interaction with the intergenic region of Sso II restriction-modification system was carried out. Seven guanine residues protected by M. Sso II from methylation with dimethylsulfate and thus probably involved in enzyme-DNA recognition were identified. Six of them are located symmetrically within the 15 bp inverted repeat inside the Sso II promoter region. The crosslinking of Sso II methyltransferase with DNA duplexes containing 5-bromo-2'-deoxyuridine (br5dU) instead of thymidine was performed. The crosslinked products were obtained in all cases, thus proving that tested thymines were in proximity with enzyme. The ability to produce the crosslinked products in one case was 2-5-fold higher than in other ones. This allowed us to imply that thymine residue in this position of the inverted repeat could be in contact with M. Sso II. Based on the experimental data, two symmetrical 4 bp clusters (GGAC), which could be involved in the interaction with M. Sso II in the DNA-protein complex, were identified. The model of M. Sso II interaction with its own promoter region was proposed.     

Identification of the nucleotide sequences specific for the 5'-flanking regions of the genes regulated by glucocorticoids by a computer analysis method

Nucleotide sequences of 5'-flanking regions of 11 glucocorticoid-regulated genes and 14 genes non-regulated by these hormones were studied using context computer analysis. Consensus TGTTCT, previously found in DNA fragments protected by glucocorticoid-receptor complexes from DNAase I digestion, was shown to be nonspecific for glucocorticoid-regulated genes. However, the analysis of sequences flanking the TGTTCT consensus has revealed that only glucocorticoid-regulated genes contain four regularly distributed cytosine residues, one of them belonging to TGTTCT consensus. Three of cytosine residues are separated by 8-10 bp, which provides their close neighbourhood at one side of DNA double helix; the fourth extreme cytosine residue is located 6 bp from the nearest one and therefore, the complementary guanine residue is adjacent to the consensus in DNA helix. It is suggested that the consensus itself and two flanking cytosine and one guanine residues form a specific site for the interaction with glucocorticoid-receptor complex.     

Demethylation of thymine residues affects DNA cleavage by endonucleases but not sequence recognition by drugs.

The 5-methyl group of thymidine residues protrudes into the major groove of double helical DNA. The structural influence of this exocyclic substituent has been examined using a PCR-made 160 bp fragment in which thymidine residues were replaced with uridine residues. We show that the dT-->dU substitution and the consequent deletion of the methyl group affects the cleavage of DNA by deoxyribonuclease I and micrococcal nuclease. Analysis of the DNase I cleavage sites, in terms of di and trinucleotides, indicates that homopolymeric tracts of d(AT) become significantly more susceptible to DNase I cleavage when uridine is substituted for thymidine residues. The results indicate that removal of the thymidine methyl groups from the major groove at AT tracts induces structural perturbations that transmit into the opposite minor groove, where they can be detected by endonuclease probing. In contrast, DNase I footprinting experiments with different mono and bis-intercalating drugs reveal that dT-->dU substitution does not markedly affect sequence-specific drug-DNA recognition in the minor or major groove of the double helix. The consequences of demethylation of thymidine residues are discussed in terms of changes in the minor groove width connected to variations in the flexibility of DNA and the intrinsic curvature associated with AT tracts. The study identifies the methyl group of thymine as an important molecular determinant controlling the width of the minor groove and/or the flexibility of the DNA.     

Photosensitized splitting of thymine dimers in DNA by gene 32 protein from phage T 4

The binding of denatured DNA to the protein coded by gene 32 of phage T 4 is accompanied by a quenching of the fluorescence of the protein tryptophyl residues. Gene 32 protein also binds to UV-irradiated DNA and photosensitizes the splitting of thymine dimers. Thymine bases are regenerated by this photosensitized reaction both in double stranded and in heat denatured DNA. No photosensitized splitting of thymine dimers is observed when the complex formed by gene 32 protein with UV-irradiated DNA is dissociated at high ionic strength. These results are discussed with respect to the possible stacking interaction of tryptophyl residues of gene 32 protein with bases in single stranded DNA.     

Non-enzymatic DNA methylation by S-adenosylmethionine results in the formation of minor thymine residues and 5-methylcytosine from cytosine

It was found that nonenzymatic DNA methylation proceeds in water solution in the presence of S-adenosylmethionine (AdoMet). The main reaction products are thymine and 5-methylcytosine residues. It was shown that labelled thymine residues are formed also upon DNA incubation in the presence of [methyl-14C]methionine as well as [methyl-14C]cobalamine. Only cytosine reacts with AdoMet resulting in thymine production. AdoMet may be a potential mutagen that induces GC----AT transitions during DNA replication in the cell.