An oligopurine sequence bias occurs in eukaryotic viruses.

Twenty four DNA and RNA viral nucleotide sequences, comprising over 346 kilobases, have been analyzed for the occurrence of strings of contiguous purine or pyrimidine residues. On average strings greater than or equal to 10 contiguous purines or pyrimidines are found three and a half times more frequently than would be expected for a random distribution of bases. Detailed analysis of the 172 kilobase Epstein-Barr viral sequence shows that the bias in favor of contiguous purine residues increases with the length of the purine string. These findings are similar to those seen for genomic DNA from higher eukaryotes. In contrast no overrepresentation of oligopurine or oligopyrimidine strings is observed in 52 kilobases from eight bacteriophage and E. coli DNA sequences.     

Local sequence dependence of rate of base replacement in mammals.

I have analysed the local sequence context of base replacement changes in 78 processed pseudogenes. Transversions occur more often than transitions in a ratio of 3.37 to 1, and G:C is replaced 1.4 times more frequently than A:T. In addition, the bases to the 5' and 3' of the mutating base also influence the rate at which bases change, purine:pyrimidine and pyrimidine:purine pairs changing 1.2 times as fast as purine:purine and pyrimidine:pyrimidine pairs. I discuss implications of this for the mechanism of DNA polymerization in mammals.     

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.     

Origin of ultraviolet damage in DNA

A novel ultraviolet (u.v.) footprinting technique has been used to analyze the formation of u.v. photoproducts at 250 bases of a 5 S rRNA gene under conditions where the gene is either double or single-stranded. Because many more types of u.v. damage can be detected by the u.v. footprinting technique than has been previously possible, we have been able to examine in detail why certain bases in DNA are damaged by u.v. light while others are not. Our measurements demonstrate that the ability of u.v. light to damage a given base in DNA is determined by two factors, the sequence of the DNA in the immediate vicinity of the photoproduct, and the flexibility of the DNA at the site of the photoproduct. For pyrimidines, the predominant photoreaction in double-stranded DNA involves covalent dimerization between adjacent pyrimidine residues. Dimerization is much easier in melted DNA because the geometrical changes required for adjacent pyrimidine residues to dimerize are easier in single-stranded DNA. The absorption of a u.v. photon cannot simultaneously induce the geometrical changes required for adjacent pyrimidines or other bases to dimerize with one another. Rather, upon the absorption of a u.v. photon, only those thermally excited bases that are in a geometry capable of easily forming a photodimer during excitation, can photoreact. In contrast to adjacent pyrimidines, non-adjacent pyrimidines (pyrimidines flanked on either side by a purine) do not readily form u.v. photoproducts in double-stranded DNA. Because photoreactions at non-adjacent pyrimidine residues are greatly enhanced in single-stranded DNA, their failure to form in double-helical DNA is attributed to torsional constraints imposed by the double helix which make it difficult for non-adjacent pyrimidines to adopt a geometry necessary for photoreaction. Although purines are believed to be resistant to u.v. damage, our measurements demonstrate that at moderate u.v. dosages purines which are flanked on their 5' side by two or more contiguous pyrimidines readily form u.v. photoproducts in double-stranded DNA. Flanking pyrimidines appear to activate purine photoreactions by transferring triplet excitation energy to the purine. Melting of the DNA helix greatly inhibits the ability of flanking pyrimidines to activate purine photoreactions, presumably by disrupting intimate orbital overlap required for triplet transfer.     

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.     

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.     

Neuroblastoma Growth Factors Derived from Neurofibroma (NF1): Participation of Uridine in a Neuroblastoma Growth

Abstract: Human glioma cell extracts were found to elicit a marked growth-promoting activity on human neuroblastoma cells. This activity was also detected in the extracts of neurofibroma type 1 (NF1; von Recklinghausen neurofibromatosis) comprising aberrant Schwann cell growth. The purified substance from the NF1 extracts by HPLC on ODS columns was identical to a pyrimidine nucleoside, uridine, the chemical structure of which was identified by gas chromatography-mass spectrometry. The authentic uridine showed a strong growth-promoting activity on human neuroblastoma cells. Other purine or pyrimidine nucleotides, their derivatives, and ribose sources for their syntheses were employed to test the activity; a purine nucleoside, adenosine, showed a stronger activity than uridine. The current study raises the possibility that human neuroblastoma cells may be affected by dysfunctions of the de novo pathway of both purine and pyrimidine nucleotide biosyntheses.     

Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region

We isolated over 20 phage clones carrying the ornithine transcarbamylase (OTC) [EC 2.1.3.3] gene, from two independently constructed human genomic DNA libraries, using as probes either a rat OTC cDNA or several nuclear DNA fragments derived from some of these isolated clones. These clones, classified into 10 different groups, overlapped and spanned a region of more than 85 kilobase pairs of the human genomic DNA. Restriction mapping and Southern blot analyses demonstrated that one of the clones covers the 5'-end region of the OTC gene. We sequenced the 5'-end region of the OTC gene and found that it covered 665 base pairs of the 5'-flanking region, the complete first exon and a part of the first intron (150 base pairs). In the 5'-flanking region, there were two pairs of putative CAAT and TATA boxes and one enhancer core-like sequence, GTGGAAAG. The first exon contained a coding region for most of the OTC presequence, i.e. 26 out of 32 amino acid residues of the presequence, including the initiation methionine.     

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.     

Docking simulation with a purine nucleoside specific homology model of deoxycytidine kinase, a target enzyme for anticancer and antiviral therapy

5'-Phosphorylation, catalyzed by human deoxycytidine kinase (dCK), is a crucial step in the metabolic activation of anticancer and antiviral nucleoside antimetabolites, such as cytarabine (AraC), gemcitabine, cladribine (CdA), and lamivudine. Recently, crystal structures of dCK (dCKc) with various pyrimidine nucleosides as substrates have been reported. However, there is no crystal structure of dCK with a bound purine nucleoside, although purines are good substrates for dCK. We have developed a model of dCK (dCKm) specific for purine nucleosides based on the crystal structure of purine nucleoside bound deoxyguanosine kinase (dGKc) as the template. dCKm is essential for computer aided molecular design (CAMD) of novel anticancer and antiviral drugs that are based on purine nucleosides since these did not bind to dCKc in our docking experiments. The active site of dCKm was larger than that of dCKc and the amino acid (aa) residues of dCKm and dCKc, in particular Y86, Q97, D133, R104, R128, and E197, were not in identical positions. Comparative docking simulations of deoxycytidine (dC), cytidine (Cyd), AraC, CdA, deoxyadenosine (dA), and deoxyguanosine (dG) with dCKm and dCKc were carried out using the FlexX docking program. Only dC (pyrimidine nucleoside) docked into the active site of dCKc but not the purine nucleosides dG and dA. As expected, the active site of dCKm appeared to be more adapted to bind purine nucleosides than the pyrimidine nucleosides. While water molecules were essential for docking experiments using dCKc, the absence of water molecules in dCKm did not affect the ability to correctly dock various purine nucleosides.     

Oxidative DNA base damage by the antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine)

Tirapazamine is a bioreductively activated DNA-damaging agent that selectively kills the hypoxic cells found in solid tumors. In this work, base excision repair enzymes were used to provide evidence that tirapazamine causes significant amounts of damage to both purine and pyrimidine residues in double-stranded DNA.     

The Effect of Local Nucleotides on Synonymous Codon Usage in the Honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) Genome

Using all currently predicted coding regions in the honeybee genome, a novel form of synonymous codon bias is presented that affects the usage of particular codons dependent on the surrounding nucleotides in the coding region. Nucleotides at the third codon site are correlated, dependent on their weak (adenine [A] or thyamine [T]) versus strong (guanine [G] or cytosine [C]) status, to nucleotides on the first codon site which are dependent on their purine (A/G) versus pyrimidine (C/T) status. In particular, for adjacent third and first site nucleotides, weak–pyrimidine and strong–purine nucleotide combinations occur much more frequently than the underabundant weak–purine and strong–pyrimidine nucleotide combinations. Since a similar effect is also found in the noncoding regions, but is present for all adjacent nucleotides, this coding effect is most likely due to a genome-wide context-dependent mutation error correcting mechanism in combination with selective constraints on adjacent first and second nucleotide pairs within codons. The position-dependent relationship of synonymous codon usage is evidence for a novel form of codon position bias which utilizes the redundancy in the genetic code to minimize the effect of nucleotide mutations within coding regions. [Reviewing Editor: Dr. Brian Morton]     

Nucleoside deoxyribosyltransferase-II from Lactobacillus helveticus Substrate specificity studied. Pyrimidine bases as acceptors.

The nucleoside deoxyribosyltransferase (nucleoside:purine (pyrimidine) deoxyribosyltransferase, EC 2.4.2.6) fraction catalyzing specifically the transfer of the deoxyribosyl moiety from a purine (or a pyrimidine) to a pyrimidine (or a purine) exhibits a broad specificity for the acceptor base. With a pyrimidine base as the acceptor a -OH or -SH group adjacent to the N-1 atom is essential. A substituent on position 6 hinders the reaction. On positions 4 and 5 various substituent were found to influence the reaction rate and some of them give non-competent substrates. A few anomalous cases are also discussed in relation with the role of N-3. Deoxyribonucleosides can also be obtained with non-pyrimidine rings.     

Stable sheared A.C pair in DNA hairpins

Single-residue d(Pu1NPu2) (Pu1.Pu2=G.A, G.G or A.A) hairpin loops can be stably closed by sheared purine.purine pairs. These special motifs have been found in several important biological systems. We now extend these loop-closing base-pairs to a sheared purine. pyrimidine (A.C) pair at a neutral pH condition. High-resolution NMR spectroscopy, distance geometry, and molecular dynamics methods were used to study d(GTACANCGTAC) oligomers. Numerous idiosyncratic nuclear Overhauser enhancements, especially those across the A.C base-pair between C4NH2left and right arrow AH1', C4NH2left and right arrow AH2, and CH5left and right arrow AH2 proton pairs, clearly define the novel sheared nature of the closing A.C base-pair. This novel base-pair is possibly present in several biological systems and in two single-stranded DNA aptamers selected from oligonucleotide libraries.     

The role of the polypyrimidine stretch at the SV40 early pre-mRNA 3'' splice site in alternative splicing.

We have studied the role in pre-mRNA splicing of the nucleotide sequence preceding the SV40 early region 3' splice site. Somewhat surprisingly, neither the pyrimidine at the highly conserved -3 position, nor the polypyrimidine stretch that extends from -5 to -15, relative to the 3' splice site, were found to be required for efficient splicing. Mutations that delete this region or create polypurine insertions at position -2 had no significant effects on the efficiency of SV40 early pre-mRNA splicing in vivo or in vitro. Interestingly, however, the pyrimidine content of this region had substantial effects on the alternative splicing pattern of this pre-mRNA in vivo. Mutations that increased the number of pyrimidine residues resulted in more efficient utilization of the large T antigen mRNA 5' splice site relative to the small t 5' splice site, while mutations that increased the purine content enhanced small t mRNA splicing. A possible molecular mechanism for these findings, as well as a model that proposes a role for the polypyrimidine stretch in alternative splicing, are discussed.     

Absence of demonstrable linkage of human genes for enzymes of the purine and pyrimidine salvage pathways in human-mouse somatic cell hybrids

A number of different reduced human-mouse hybrids have been analyzed for the presence of human enzymes of the purine and pyrimidine salvage pathways. Homologous mouse and human enzymes were characterized by isoelectric fractionation or gel electrophoresis, and the species of origin of the enzyme in hybrid clones was determined. Hybrids selected for one of the human enzymes, thymidine kinase, adenine phosphoribosyltransferase, or hypoxanthine phosphoribosyltransferase, were each found to contain the selected enzyme but not the other two. Neither human adenosine kinase nor human deoxycytidine (cytidine) deaminase was present in any of the hybrid clones. A human 5-nucleotidase was present in two hybrid clones containing human hypoxanthine phosphoribosyltransferase, but the genes for the two enzymes are not linked. The genes for the purine and pyrimidine salvage enzymes appear to be dispersed in the human genome.These investigations were aided by a grant from the National Cancer Institute.     

A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines

In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, but can also phosphorylate purine substrates. When the structures of the human deoxyguanosine kinase (dGK, EC 2.7.1.113) and the dNK from Drosophila melanogaster were compared, a limited number of amino acid residues were identified and proposed to be responsible for the substrate specificity. Three of these key residues in Drosophila dNK were then mutagenized and the mutant enzymes were characterized regarding their ability to phosphorylate native deoxyribonucleosides and nucleoside analogs. The mutations converted the dNK substrate specificity from predominantly pyrimidine specific into purine specific. A similar scenario could have been followed during the evolution of kinases. Upon gene duplication of the progenitor kinase, only a limited number of single amino acid changes has taken place in each copy and resulted in substrate-specialized enzymes.     

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.