The DNA sequence of the human beta-globin region is strongly biased in favor of long strings of contiguous purine or pyrimidine residues

The DNA sequence of the human beta-globin region, comprising over 67 kilobase pairs, has been analyzed for the occurrence of strings of contiguous purine or pyrimidine residues. Tracts of 10 or more contiguous residues are found 4 times more frequently than would be expected with a random distribution of bases, so that a long string occurs at an average of every 250 base pairs. A survey of six other human gene sequences, totaling 86 kilobase pairs, shows a remarkably similar result. No such overrepresentation of contiguous purine or pyrimidine residues is found in the bacteriophages lambda or T7.     

An NMR structural study of deaminated base pairs in DNA.

The structurally aberrant base pairs TG, UG and TI may occur in DNA as a consequence of deamination of 5-methylcytosine, cytosine and adenine respectively. Results of NMR spectroscopic studies are reported here for these deaminated base pairs in a model seven base pair long oligonucleotide duplex. We find that in all three cases, the DNA helix is a normal B form and both mispaired bases are intrahelical and hydrogen bonded with one another in a wobble geometry. Similarly, in all three cases, all sugars are found to be normal C2' endo in conformation. Symmetric structural perturbations are observed in the helix twist on the 3' side of the mispaired pyrimidine and on the 5' side of the mispaired purine. In all three cases, the amino group of the G residue on the 3' side of the mispaired pyrimidine shows hindered rotation. Although less thermodynamically stable than helices containing only Watson-Crick base pairs, these helices melt normally from the ends and not from the mispair outwards.     

Unusual duplex formation in purine rich oligodeoxyribonucleotides

The purine rich oligodeoxyribonucleotides 1C, d(ATGACGGAATA) and 2C, d(ATGAGCGAATA) alone exhibit highly cooperative melting transitions. Analysis of the concentration dependence of melting, and electrophoretic studies indicate that these oligomers can form an unusual purine rich offset double helix. The unusual duplex is predicted to contain four A.T, two G.C, and four G.A mismatch base pairs as well as a single A base stacked on the 3' end of each chain of the helix. Other possible models for the duplex are unlikely because they are predicted to contain many base pairs of low stability. Changing the central sequence to CGG or GGG should destabilize the duplex and this is observed. The unusual duplex of 2C is more stable than the duplex of 1C indicating that the stability of G.A base pairs is quite sensitive to the surrounding sequence. Addition of 1C and 2C to their complementary pyrimidine strands results in normal duplexes of similar stability. We feel that the unusual duplexes are significantly stabilized by the intrinsic stacking tendency of purine bases.     

Differential extension of 3' mispairs is a major contribution to the high fidelity of calf thymus DNA polymerase-alpha

The fidelity of DNA polymerase-alpha-primase from calf thymus has been analyzed by measuring mutagenesis in vitro and by site-specific nucleotide misinsertion and mispair extension. Using the phi X174 am3 DNA reversion assay errors are detected at the amber3 site only when both dATP and dCTP are significantly biased during in vitro copying reactions. Analysis of these products on DNA sequencing gels reveals pause sites due to the slow extension of mispaired 3' termini. Measurements of misinsertion rates opposite template A show that the rates of dAMP or dCMP misinsertion are similar and occur 40-50 times more rapidly than dGMP misinsertion. The rate of extension from an A:C mispair is 100- and 400-fold greater than from an A:A mispair and an A:G mispair, respectively. Nucleotide misinsertions to generate all 12 possible mispairs have been measured kinetically on phi X174 DNA templates that contain either A, C, G, or T at position 587. Misinsertion frequencies range from 1/4000 to 1/10(6) depending on the mispairs generated. Extension from all 12 different mispairs was examined by starting with oligonucleotide primers that contain different 3'-terminal mispairs. Rates of extension from mispairs are 10(3) to 10(6) times slower than from correctly paired bases. Extension frequencies were purine:pyrimidine greater than pyrimidine:pyrimidine greater than purine:purine. Lack of extension of misincorporated bases suggests the involvement of exonucleolytic proofreading to enable continued DNA synthesis and to guarantee the high fidelity of eucaryotic DNA replication.     

Molecular mechanics calculations on a triple stranded DNA involving C+.G-T and T.A+-C mismatched base triples

We have carried out molecular modeling of a triple stranded pyrimidine(Y). purine(R): pyrimidine(Y) (where ':' refers to Watson-Crick and '.' to Hoogsteen bonding) DNA, formed by a homopurine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC). Molecular mechanics calculations using NMR constraints have provided a detailed three dimensional structure of the triplex. The entire stretches of purine and the pyrimidine nucleotides have a conformation close to B-DNA. The three strands are held by the canonical C+.G:C and T.A:T hydrogen bonds. The structure also contains two mismatch C+.G-T and T.A+-C base triples which have been characterized for the first time. In the A+-C base-pair of the T.A+-C triple, both hydrogen donors are situated on the purine (A+(1N) and A+(6N)). We observe a unique hydrogen bonding interaction scheme in case of C+.G-T where one acceptor, G(60), is bonded to three donors (C+(3NH), C+(4NH2) and T(3NH)). Though the C+.G-T base triple is less stable than C+.G:C, it is significantly more stable than T.A:T. On the other hand, T.A+-C is as stable as the T.A:T base triad.     

Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.     

Pyrimidine scavenging by Mycobacterium leprae

Mycobacterium leprae incorporated exogenously supplied pyrimidines as bases or nucleosides, but not as a nucleotide, into its nucleic acids. Notably, thymine was incorporated approximately 5 times more rapidly than thymidine by both suspensions of, or intracellular M. leprae. Thymine incorporation was significantly inhibited by clofazamine and dapsone at near-pharmacological levels. Therefore, incorporation of thymine is preferable as an activity for assessing viability of M. leprae. Nucleosides were converted to nucleotides through kinases, bases through phosphoribosyltransferases. Alternatively, thymine and uracil could first be converted to nucleosides. Cytosine and uracil bases were interconvertible, and uracil alone could supply all the pyrimidine requirements of M. leprae, though conversion to the thymine base was extremely slow. Overall, pyrimidine scavenging occurs at a slower rate than, and appears not to be so important as purine scavenging in M. leprae.     

Mechanisms of DNA damage and insight into mutations by chromium(VI) in the presence of glutathione

The mechanisms of hexavalent chromium(VI) induced DNA damage were unveiled by detecting products of single- and double-stranded DNA in the presence of glutathione. The absence of a detectable hydroxyl radical in the reactions indicates that DNA damage was exclusively by hypervalent chromium species. Polyacrylamide gel electrophoresis (PAGE) experiments with 32-mer single-stranded oligonucleotide and its complementary duplex revealed cleavages largely at purine bases with significant enhancement of such cleavages in the presence of a base. Quantitative estimations of bases released by HPLC before and after enzymatic digestion with exonucleases unequivocally established the excessive release of purine bases. This release was accompanied by the concomitant formation of phosphoglycolate as characterized by liquid chromatography-mass spectrometry (LC-MS). These data connote that the preponderance DNA damage is due to an oxidation specifically at H4' of the ribose moiety leading to the formation of apurinic sites. In addition to the oxidation at H4', DNA oxidation was also initiated through H5' site as evidenced by the identification of furfural. This pathway appears to be non-selective and more abundant for ssDNA as cleavages were observed at both purine and pyrimidine bases. Finally, the detection of guanidinohydantoin as a minor product points the involvement of an oxygen activated hypervalent chromium species, perhaps a peroxo-chromium species. Both major and minor pathways lead to cleavages at purine sites for ds-DNA and are consistent with the observation that DNA cleavage was enhanced in the presence of a base. In contrast, when hydrogen peroxide was added to the reactions, random DNA cleavages were apparent indicating involvement of multiple species including a hydroxyl radical. These data pinpoint mutation mechanisms induced by chromium(VI) in the presence of glutathione due to transversion either by inserting the wrong bases opposite to the apurinic sites during replication or by purine-purine mismatch.     

离子注入诱变莲花突变体分子机理的初步研究

低能离子注入技术作为生物物理诱变的一种新型技术, 在园艺植物育种方面具有很大的应用潜力, 但其诱变的分子机理目前知之甚少。文章对Fe+ 离子注入诱变的白洋淀红莲(Nelumbium speciosum Willd)突变体及其对照的基因组进行RAPD研究, 并将突变体和对照在辐射敏感位点的条带进行克隆测序及DNA序列分析。在已优化好的RAPD体系下扩增, 从110条随机引物中筛选出了10条可以稳定扩增出显著特异条带的引物, 引物多态性为9.09%。将这10条引物扩增出的辐射敏感位点的条带进行克隆测序, 并进行序列比对。结果显示: 突变体的总碱基突变频率为0.87%, 6个突变体的碱基突变频率存在着差异; 碱基突变类型包括碱基的颠换、转换、缺失、插入, 在检测到的159个碱基突变中, 单碱基置换的频率(61.01%)高于碱基插入或者缺失的频率(38.99%), 在碱基置换中, 转换的频率(44.65%)是颠换频率(16.35%)的2.7倍, 其中C/T之间的转换所占比例最大, A→G和A→T也具有较高的替换频率; 构成DNA的4种碱基均可以被离子束辐照诱变发生变异, 除了没有C→G的置换外, 每一种碱基都可以被其他的几种碱基所置换, 但是胸腺嘧啶(T)具有较高的辐射敏感性。通过对碱基突变位点周边序列的分析发现, 嘌呤突变位点的周围嘌呤碱居多, 嘧啶突变位点的周围嘧啶碱居多。研究结果为揭示低能离子注入诱变作用分子机理提供了依据。     

Breaking pseudo-twofold symmetry in the poliovirus 3'-UTR Y-stem by restoring Watson-Crick base pairs

The previously described NMR structure of a 5'-CU-3'/5'-UU-3' motif, which is highly conserved within the 3'-UTR Y-stem of poliovirus-like enteroviruses, revealed striking regularities of the local helix geometry, thus retaining the pseudo-twofold symmetry of the RNA helix. A mutant virus with both pyrimidine base pairs changed into Watson-Crick replicated as wild type, indicating the functional importance of this symmetry relation in viral RNA replication. Here we investigated the effect of changing only one of the two pyrimidine base pairs to Watson-Crick. We determined the NMR structures of two Y-stem variants: one containing the 5'-CU-3'/5'-AU-3' motif, which has been found in wild-type virus isolates as well, and the other containing a 5'-CU-3'/5'-UG-3' motif, which is not present in any enterovirus sequenced to date. Both structures show single pyrimidine mismatches with intercalated bases. In the 5'-CU-3'/5'-AU-3' motif a C-U Watson-Crick-type base pair is formed that retains the pseudo-twofold symmetry, while in the 5'-CU-3'/5'-UG-3' motif a single asymmetric U-U mismatch breaks the twofold symmetry. Surprisingly, for the nonnatural variant no effect of the single base-pair replacement was observed on polioviral RNA replication using an in vitro replicon assay.     

The nucleotide sequence recognised by the Escherichia coli D type I restriction and modification enzyme.

A type I restriction endonuclease from a new isolate of Escherichia coli (E. coli E166) has been purified and characterised. The enzyme, EcoD, has a recognition sequence similar in overall structure to the previously determined type I enzyme sequences, an exception being that it is degenerate. The sequence is 5'-T-T-A-N-N-N-N-N-N-N-G-T-C-Y-3' 3'-A-A-T-N-N-N-N-N-N-N-C-A-G-R-5' where Y is a pyrimidine, R is a purine and N can be any nucleotide. The enzyme methylates adenosyl residues in both strands of the DNA that are separated by ten base pairs, suggesting that the enzyme interacts with DNA along one face of the helix making contacts in two successive major grooves.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).     

Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus

Lactobacillus helveticus contains two types of N-deoxyribosyltransferases: DRTase I catalyzes the transfer of 2'-deoxyribose between purine bases exclusively whereas DRTase II is able to transfer the 2'-deoxyribose between two pyrimidine or between pyrimidine and purine bases. An Escherichia coli strain, auxotrophic for guanine and unable to use deoxyguanosine as source of guanine, was constructed to clone the corresponding genes. By screening a genomic bank for the production of guanine, the L. helveticus ptd and ntd genes coding for DRTase I and II, respectively, were isolated. Although the two genes have no sequence similarity, the two deduced polypeptides display 25.6% identity, with most of the residues involved in substrate binding and the active site nucleophile Glu-98 being conserved. Overexpression and purification of the two proteins shows that DRTase I is specific for purines with a preference for deoxyinosine (dI) > deoxyadenosine > deoxyguanosine as donor substrates whereas DRTase II has a strong preference for pyrimidines as donor substrates and purines as base acceptors. Purine analogues were substrates as acceptor bases for both enzymes. Comparison of DRTase I and DRTase II activities with dI as donor or hypoxanthine as acceptor and colocalization of the ptd and add genes suggest a specific role for DRTase I in the metabolism of dI.     

Importance of minor groove functional groups for the stability of DNA duplexes

Eight oligonucleotide duplexes have been prepared with four pairs of selected complementary pairs of native/analogue heterocyclic bases incorporated at a selected test site. The base pairs vary in the nature of their functionality in the minor groove. Each pair has a minor groove purine amino group present or absent, and correspondingly has a minor grove pyrimidine carbonyl present or absent. Loss of duplex stability is most notable when the minor groove pyrimidine carbonyl is absent although in other respects normal Watson-Crick hydrogen bonding is maintained in these sequences. These differences in stability are discussed in terms of possible variations in minor groove hydration.     

Nucleobase analogs for degenerate hybridization devised through conformational pairing analysis

A conformational pairing analysis was used to devise nucleobase analogs capable of forming nonselective and energetically favorable base pairs opposite either the purine or the pyrimidine constituents of nucleic acids. 5-methylisocytosine and isoguanine were conceived as a degenerate pyrimidine and a degenerate purine, respectively. Data from previous DNA duplex melting experiments verified that the analogs can act as degenerate nucleobases as hypothesized. Isoguanine also formed unusually stable base pairs with guanine. A quantitative PCR assay yielding equivalent results across hepatitis C virus (HCV) subtypes was created with this system, despite the use of a single probe targeted to a polymorphic region. Amplification curves using probes with 5-methylisocytosine or isoguanine opposite appropriate ambiguous target positions exhibited more signal than curves from similar probes containing common degenerate nucleobase hypoxanthine.     

A theoretical study oh the effect of "bound" water on the proton chemical shifts of the nucleic acid bases.

Computations are performed on the proton chemical shifts due to hydrogen bonding between the purine and pyrimidine bases of the nucleic acids and water molecules of their first hydration shell. The water molecules should produce measurable shifts essentially for protons of the bases located close to the site of interaction. For the imino protons of the bases G-N1H and U-N3H participating in hydrogen bonding, the calculated delta delta is larger for the interaction of a base with a complementary base than for its interaction with water. Base pairing will thus produce a downfield shift in water but the measured delta delta due to pairing in this solvent will be smaller than in an inert solvent. Also, the chemical shift difference between G-N1H and U-N3H in water will be larger if the molecules are engaged in pairs than if they are not.     

Fluorescence-excitation spectra of acridine orange-DNA and -RNA systems

Summary The absorption, fluorescence and fluorescence-excitation spectra were investigated in free AO, AO-DNA and AO-RNA systems. In the case of Complex I, AO seems to be metachromatically bound to the two kinds of binding sites, phosphates neighboring purine bases and those neighboring pyrimidine bases, and becomes non-fluorescent.In the case of Complex II, the AO dimer originally present in solution dissociates into monomers and the monomer is intercalated between base pairs, resulting in the enhancement of fluorescence emission. These are suggested by the difference between the absorption band and the fluorescence-excitation spectrum normalized at the maximum to the absorption. The electronic processes of optical behaviours in free and bound AO are discussed on the basis of the emission properties at 77 °K.     

Effects of base sequence on the loop folding in DNA hairpins

High-resolution NMR and UV-melting experiments have been used to study the hairpin formation of partly self-complementary DNA fragments in an attempt to derive rules that describe the folding in these molecules. Earlier experiments on the hexadecanucleotide d(ATCCTA-TTTT-TAGGAT) had indicated that within the loop of four thymidines a wobble T-T pair is formed (Blommers et al., 1987). In the present paper it is shown that if the first and the last thymines of the intervening sequence are replaced by complementary bases, sometimes base pairs can be formed. Thus for the intervening sequences -CTTG- and -TTTA- with the pyrimidine in the 5'-position and the purine in the 3'-position, a base pair is formed leading to a loop consisting of two residues. For the intervening sequences -GTTC- and -ATTT- with the purine in the 5'-position and the pyrimidine in the 3'-position, this turns out not to be the case. It was found that it made no difference when the four-membered sequence was closed by a G-C base pair or an A-T base pair. Replacement of the two central thymidine residues by the more bulky adenine residues limits the hairpin to a four-membered loop scheme. Very surprisingly, it was found from 2D NOE experiments that the T-A base pair, formed in the loop consisting of the -TTTA- sequence, is a Hoogsteen pair. It is argued that the pairing of the bases in this scheme may facilitate the formation of a loop of two residues, since the distance of the C1' atoms in this base pair is 8.6 A instead of 10.4 A found in the canonical Watson-Crick base pair. Combination of the data obtained for the series of DNA fragments studied shows that the results can be explained by a simple, earlier proposed, loop folding principle which assumes that the folding of the four-membered loop is dictated by the stacking of the double-helical stem of the hairpin.     

Estimation of the lipophilicity of anti-HIV nucleoside analogues by determination of the partition coefficient and retention time on a Lichrospher 60 RP-8 HPLC column

There is a close linear correlation between the log partition coefficient (Pa) of a series of 2'-deoxyriboside (dR), 2',3'-didehydro-3'-dideoxyriboside (ddeR), 2',3'-dideoxyriboside (ddR), 3'-fluoro-2',3'-dideoxyriboside (FddR) and 3'-azido-2',3'-dideoxyriboside (AzddR) derivatives of uracil, cytosine, thymine, guanine, adenine and 2,6-diaminopurine and their retention times (Rt) on a Lichrospher 60 RP-8 HPLC column (correlation coefficient r greater than 0.970). Within each class of compounds the following order of increasing lipophilicity was noted: dR less than ddeR less than ddR less than FddR less than AzddR. A straight-forward structure-lipophilicity relationship for both base and sugar modified purine and pyrimidine 2',3'-dideoxynucleosides could be delineated.