The dgt gene of Escherichia coli facilitates thymine utilization in thymine-requiring strains.

The Escherichia coli dGTP triphosphohydrolase (dGTPase) encoded by the dgt gene catalyses the hydrolysis of dGTP to deoxyguanosine and triphosphate. The recent discovery of a mutator effect associated with deletion of dgt indicated participation of the triphosphohydrolase in preventing mutagenesis. Here, we have investigated the possible involvement of dgt in facilitating thymine utilization through its ability to provide intracellular deoxyguanosine, which is readily converted by the DeoD phosphorylase to deoxyribose-1-phosphate, the critical intermediate that enables uptake and utilization of thymine. Indeed, we observed that the minimal amount of thymine required for growth of thymine-requiring (thyA) strains decreased with increased expression level of the dgt gene. As expected, this dgt-mediated effect was dependent on the DeoD purine nucleoside phosphorylase. We also observed that thyA strains experience growth difficulties upon nutritional shift-up and that the dgt gene facilitates adaptation to the new growth conditions. Blockage of the alternative yjjG (dUMP phosphatase) pathway for deoxyribose-1-phosphate generation greatly exacerbated the severity of thymine starvation in enriched media, and under these conditions the dgt pathway becomes crucial in protecting the cells against thymineless death. Overall, our results suggest that the dgt-dependent pathway for deoxyribose-1-phosphate generation may operate under various cell conditions to provide deoxyribosyl donors.

Apoptosis in CADASIL: an in vitro study of lymphocytes and fibroblasts from a cohort of Italian patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease affecting vascular smooth muscle cells of nearly all tissues. Clinical manifestations mainly concern the central nervous system with repeated TIA/stroke, migraine, psychiatric disturbances, and cognitive decline. Minor findings have been reported in muscle, nerve, and skin. CADASIL is due to NOTCH3 gene mutations. This gene has been identified as an up-regulator of c-FLIP, an inhibitor of Fas-ligand-induced apoptosis. The aim of this study was to assess the involvement of oxidative stress-induced apoptosis in cells from 16 Italian CADASIL patients. Peripheral blood lymphocytes (PBLs) and fibroblasts from CADASIL patients were exposed to 2-deoxy-D-ribose (dRib), which induces apoptosis by oxidative stress. Apoptosis was analyzed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy for caspase-3 activation, phosphatidylserine exposure and mitochondrial membrane depolarization. PBLs and fibroblasts from CADASIL patients showed a significantly higher response to dRib-induced apoptosis than those of controls. PBLs from CADASIL patients also showed a significantly higher percentage of apoptotic cells than PBLs from controls, even when cultured without dRib. The greater susceptibility of PBLs and fibroblasts from CADASIL patients to dRib-induced apoptosis suggests that NOTCH3 mutations are an important apoptotic trigger. Since PBLs from patients showed higher levels of apoptosis even in the absence of an apoptotic stimulus, cells from CADASIL patients appear to be physiologically prone to apoptotic cell death.

2'-deoxyribonolactone lesion produces G->A transitions in Escherichia coli.

2'-deoxyribonolactone (dL) is a C1'-oxidized abasic site damage generated by a radical attack on DNA. Numerous genotoxic agents have been shown to produce dL including UV and gamma-irradiation, ene-dye antibiotics etc. At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides. We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay. The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZalpha gene by a change in plaque color. In E.coli JM105 the dL-induced reversion frequency was 4.7 x 10(-5), similar to that of the classic abasic site 2'-deoxyribose (dR). Here we report that a dL residue in a duplex DNA codes mainly for thymidine. The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains. We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo.

Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants.

This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4' alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.

Solution structure of an oligonucleotide containing an abasic site: evidence for an unusual deoxyribose conformation.

The antitumor antibiotic bleomycin causes two major lesions in the deoxyribose backbone of DNA: formation of 4'-keto abasic sites and formation of strand breaks with 3'-phosphoglycolate and 5'-phosphate ends. As a model for the 4'-keto abasic site, we have characterized an abasic site (X) in d(CCAAAGXACTGGG).d(CCCAGTACTTTGG) by two-dimensional NMR spectroscopy. A total of 475 NOEs and 101 dihedral angles provided the restraints for molecular modeling. Four unusual NOEs were observed between each anomer of the abasic site and the neighboring bases. In addition, four coupling constants for adjacent protons of the deoxyribose of both the alpha and beta anomers of the abasic site were observed. The modeling suggests that for both anomers the abasic site is extrahelical, without significant distortion of the backbone opposite the lesion. The coupling constants further allowed assignment of an unusual sugar pucker for each anomer. The unique position of the abasic site in our structural model for each anomer is discussed in terms of repair of such lesions in vivo.

Experimental and computational studies of the G[UUCG]C RNA tetraloop.

In prokaryotic ribosomal RNAs, most UUCG tetraloops are closed by a C-G base-pair. However, this preference is greatly reduced in eukaryotic rRNA species where many UUCG tetraloops are closed by G-C base-pairs. Here, biophysical properties of the C[UUCG]G and G[UUCG]C tetraloops are compared, using experimental and computational methods. Thermal denaturation experiments are used to derive thermodynamic parameters for the wild-type G[UUCG]C tetraloop and variants containing single deoxy substitutions in the loop. A comparison with analogous experiments on the C[UUCG]G motif shows that the two RNA species exhibit similar patterns in response to the substitutions, suggesting that their loop structures are similar. This conclusion is supported by NMR data that suggest that the essential UUCG loop structure is maintained in both tetraloops. However, NMR results show that the G[UUCG]C loop structure is disrupted prior to melting of the stem; this behavior is in contrast to the two-state behavior of the C[UUCG]G molecule. Stochastic dynamics simulations using the GB/SA continuum solvation model, run as a function of temperature, show rare conformational transitions in several G[UUCG]C simulations. These results lead to the conclusion that substitution of a G-C for a C-G closing base-pair increases the intrinsic flexibility of the UUCG loop.

Experimental and theoretical studies of the effects of deoxyribose substitutions on the stability of the UUCG tetraloop.

Experimental and theoretical thermodynamic studies of the consequences of 2'-hydroxyl substitution in the RNA UUCG tetraloop show distinct position dependence consistent with the diverse structural contexts of the four-loop ribose hydroxyls in this motif. The results suggest that even for simple substitutions, such as the replacement of the ribose hydroxyl (2'-OH) with hydrogen (2'-H), the free energy change reflects a complex interplay of hydrogen bonding and solvation effects and is influenced by the intrinsic pucker preferences of the nucleotides. Furthermore, theoretical studies suggest that the effect of these mutations in the single-strand state is sequence dependent, in contrast to what is commonly assumed. Free energy perturbation simulations of ribose-deoxyribose mutations in a single-strand dodecamer and in trinucleotide models suggest that in the denatured state, the magnitude of the free energy change for deoxyribose substitutions is determined to a larger extent by the identity of the nucleotide (A, C, G or U) rather than its structural context. Single-strand mutational effects must be considered when interpreting mutational studies in molecular terms.

An A-type double helix of DNA having B-type puckering of the deoxyribose rings.

DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of trifluoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(C(n)G(n)) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base-pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition.

Solution structure and dynamics of the A-T tract DNA decamer duplex d(GGTAATTACC)2: implications for recognition by minor groove binding drugs.

The structure of the DNA decamer duplex d(GGTAATTACC)(2) has been determined using NMR distance restraints and molecular dynamics simulations of 500 ps to 1 ns in aqueous solution at 300 K. Using both canonical A and canonical B starting structures [root-mean-square deviation (RMSD) 4.6 A; 1 A=10(-10) m], with and without experimental restraints, we show that all four simulations converge to a similar envelope of final conformations with B-like helical parameters (pairwise RMSD 1.27-2.03 A between time-averaged structures). While the two restrained simulations reach a stable trajectory after 300-400 ps, the unrestrained trajectories take longer to equilibrate. We have analysed the dynamic aspects of these structures (sugar pucker, helical twist, roll, propeller twist and groove width) and show that the minor groove width in the AATT core of the duplex fluctuates significantly, sampling both wide and narrow conformations. The structure does not have the highly pre-organized narrow minor groove generally regarded as essential for recognition and binding by small molecules, suggesting that ligand binding carries with it a significant component of 'induced-fit'. Our simulations show that there are significant differences in structure between the TpA step (where p=phosphate) and the ApA and ApT steps, where a large roll into the major groove at the TpA step appears to be an important factor in widening the minor groove at this position.

Mechanisms and pathways from recent deoxysugar biosynthesis research.

In the past few years, there have been many important advances in our understanding of the biosynthesis of deoxysugars. Mechanistic studies have shed light on how enzymes can cleave C-O bonds, epimerize the configuration of substituents and reduce keto groups to make deoxysugars. Exciting progress has also been made in our comprehension of the genetics of deoxysugar biosynthesis in antibiotics. All this information is important for potential medical and biotechnological applications, such as drug discovery based on combinatorial biology.

DNA synthesis arrest at C4'-modified deoxyribose residues.

Many genotoxic agents form base lesions that inhibit DNA polymerases. To study the mechanism underlying termination of DNA synthesis on defective templates, we tested the capacity of a model enzyme (Klenow fragment of Escherichia coli DNA polymerase I) to catalyze primer elongation across a series of C4' deoxyribose derivatives. A site with inverted C4' configuration or two different C4' deoxyribose adducts were introduced into the backbone of synthetic templates without modifying the chemistry of the corresponding bases. Inverted deoxyribose moieties may arise in cellular DNA as a product of C4' radical attack. We found that DNA synthesis by the Klenow polymerase was arrested transiently at the C4' inversion and was essentially blocked at C4' deoxyribose adducts. Major termination sites were located one position downstream of a C4' selenophenyl adduct and immediately 3' to or opposite a C4' pivaloyl adduct. Primer extension studies in the presence of single deoxyribonucleotides showed intact base pairing fidelity opposite all three C4' variants regardless of whether the Klenow fragment or its proofreading-deficient mutant was tested. These results imply that the coding ability of template bases is maintained at altered C4' deoxyribose moieties. However, their capacity to impede DNA polymerase progression indicates that backbone distortion and steric hindrance are important determinants of DNA synthesis arrest on damaged templates. The strong inhibition by C4' adducts suggests a potential target for new chemotherapeutic strategies.

Addition of deoxyribose to guanine and modified DNA based by Lactobacillus helveticus trans-N-deoxyribosylase.

The use of bacterial trans-N-deoxyribosylase was evaluated as an alternative method for deoxyribosylation in the synthesis of deoxyribonucleosides containing potentially mutagenic adducts. A crude enzyme preparation was isolated from Lactobacillus helveticus and compared to Escherichia coli purine nucleoside phosphorylase. trans-N-deoxyribosylase was more regioselective than purine nucleoside phosphorylase in the deoxyribosylation of Gua at the N9 atom, as compared to N7, as demonstrated by NMR analysis of the product. 5,6,7,9-Tetrahydro-7-acetoxy-9-oxoimidazo[1,2-a]purine was efficiently deoxyribosylated by trans-N-deoxyribosylase but not at all by purine nucleoside phosphorylase. Other substrates for trans-N-deoxyribosylase were N2-(2-oxoethyl)Gua, pyrimido[1,2-a]purin-10(3H)-one, 1,N2-epsilon-Gua, N2,3-epsilon-Gua, 3,N4-epsilon-Cyt, 1,N6-epsilon-Ade, C8-methylGua, and C8-aminoGua, most of which gave the desired isomer (bond at the nitrogen corresponding to N9 in Gua) in good yield. Neither N7-alkylpurines nor C8-(arylamino)-substituted guanines were substrates. The approach offers a relatively convenient method of enzymatic preparation of many carcinogen-DNA adducts at the nucleoside level, for either use as standards or incorporation into oligonucleotides. trans-N-deoxyribosylase can also be used to remove deoxyribose from modified deoxyribonucleosides in the presence of excess Cyt.

Crude extracts of hepatoprotective plants, Solanum nigrum and Cichorium intybus inhibit free radical-mediated DNA damage.

The presence of plant extracts of Solanum nigrum and Cichorium intybus in the reaction mixture containing calf thymus DNA and free radical generating system protect DNA against oxidative damage to its deoxyribose sugar moiety. The effect was dependent on the concentration of plant extracts. However, the effect of Cichorium intybus was much pronounced as compared to the effect of Solanum nigrum. These studies suggest that the observed hepatoprotective effect of these crude plant extracts may be due to their ability to suppress the oxidative degradation of DNA in the tissue debris.

Crystal and molecular structure of a DNA fragment: d(CGTGAATTCACG).

The crystal structure of the dodecanucleotide d(CGTGAATTCACG) has been determined to a resolution of 2.7 A and refined to an R factor of 17.0% for 1532 reflections. The sequence crystallizes as a B-form double helix, with Watson-Crick base pairing. This sequence contains the EcoRI restriction endonuclease recognition site, GAATTC, and is flanked by CGT on the 5'-end and ACG on the 3'-end, in contrast to the CGC on the 5'-end and GCG on the 3'-end in the parent dodecamer d(CGCGAATTCGCG). A comparison with the isomorphous parent compound shows that any changes in the structure induced by the change in the sequence in the flanking region are highly localized. The global conformation of the duplex is conserved. The overall bend in the helix is 10 degrees. The average helical twist values for the present and the parent structures are 36.5 degrees and 36.4 degrees, respectively, corresponding to 10 base pairs per turn. The buckle at the substituted sites are significantly different from those seen at the corresponding positions in the parent dodecamer. Step 2 (GpT) is underwound with respect to the parent structure (27 degrees vs 36 degrees) and step 3 (TpG) is overwound (34 degrees vs 27 degrees). There is a spine of hydration in the narrow minor groove. The N3 atom of adenine on the substituted A10 and A22 bases are involved in the formation of hydrogen bonds with other duplexes or with water; the N3 atom of guanine on G10 and G22 bases in the parent structure does not form hydrogen bonds.

Hexagonal crystal structure of the A-DNA octamer d(GTGTACAC) and its comparison with the tetragonal structure: correlated variations in helical parameters.

The alternating DNA octamer d(GTGTACAC) has been grown in a novel hexagonal crystal form. The structure has been determined and refined to a 2-A resolution, with 51 water molecules. The A-DNA conformation is a variant of that observed for the tetragonal form of the same sequence (Jain et al., 1989) containing a bound spermine. The crystals belong to the space group P6(1)22, a = b = 32.40 A and c = 79.25 A, with one strand in the asymmetric unit. The new hexagonal structure was solved by rotation and translation searches in direct space and refined to a final R value of 12.7% by using 1561 unique reflections greater than 1.5 sigma (I). The electron density clearly shows that the penultimate A7 sugar had flipped into the alternative C2'-endo pucker. This dent in the molecule can be attributed to close intermolecular contacts. In contrast, in the tetragonal structure, the DNA is distorted in the central TA step, where the A5 backbone bonds C4'-C5' and O5'-P assume trans conformations. The hexagonal double helix more closely resembles the fiber diffraction A-DNA, compared to the tetragonal form. For instance, the tilt angle is higher (16 degrees vs 10 degrees), which is correlated with a larger displacement from the helix axis (3.5 vs 3.3), a lower rise per residue (2.9 vs 3.2), and a smaller major-groove width (6.1 vs 8.7), thus indicating that the variations in these global helical parameters are correlated. The propeller twist angles in both forms are higher for the G-C base pairs (15.3 degrees, 12.14 degrees) than for the A-T base pairs (10.8 degrees, 9.1 degrees), which is the reverse of the expected order. Unlike the tetragonal structure, the hexagonal crystal structure interestingly does not contain a bound spermine molecule. Our analysis reveals that the conformational differences between the tetragonal and hexagonal forms are not entirely due to the spermine binding, and crystal packing seems to play an important role.

Chemical determination of free radical-induced damage to DNA.

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

Multiple DNA repair activities for 3'-deoxyribose fragments in Escherichia coli.

Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolaldehyde, PGA) from the 3' termini of a synthetic DNA substrate. The major such activities are the main bacterial apurinic endonucleases, exonuclease III and endonuclease IV. In a double mutant deficient in both of these oxidation repair enzymes, Mg++-dependent 3'-PGA diesterase was detected at 3% the level found in wild-type bacteria. Gel filtration fractionated this residual diesterase activity into two peaks of Mr 40,000-52,000 (Pool A) and Mr 22,000-30,000 (Pool B) with differing abilities to remove 3'-phosphates from DNA. These multiple repair activities were resolved in 3'-PGA diesterase activity gels. The exonuclease III and endonuclease IV bands were identified using the purified proteins and by their specific absence from strains defective for the respective structural genes. Gel filtration Pool B yielded two activity bands of apparent Mr 25,000 and 28,000, but Pool A did not form a new band in the activity gels. Incubation of activity gels in different transition metals or boiling of the samples before electrophoresis also served to distinguish the various activities. The possible identities of the novel E. coli 3'-PGA diesterases and the importance of multiple repair enzymes for 3' damages are discussed.