Guanosine Residues Research Articles

FR900482, a close cousin of mitomycin C that exploits mitosene-based DNA cross-linking.

The class of antitumor antibiotics that includes FR900482 has a very close structural analogy to the mitomycins, one of which, mitomycin C, has been in widespread clinical use for more than 20 years. Like mitomycin C, these antitumor antibiotics are reductively activated in vivo and covalently cross-link DNA as a result of activity of the mitosene moiety generated on reduction. Owing to differences in structure and the attendant mechanistic differences in bioreductive activation between the mitomycins and FR900482, FR900482 does not produce an adventitious superoxide radical anion during reductive activation and thus does not exhibit oxidative strand scission of DNA. It is postulated that the low clinical toxicity of FR900482 relative to mitomycin C is a direct manifestation of the mechanistic differences of bioreductive activation leading to the highly reactive DNA cross-linking mitosenes. Using Fe(II)-EDTA footprinting, we showed that the two natural products FR900482 (1) and dihydro, FR66979 (3), and the semi-synthetically derived triacetate FK973 (2), display remarkable selectivity for 5' deoxy-CG sequences of DNA, and that this selectivity is abolished upon deletion of the exocyclic N2 amine of either participating guanosine residue. In addition, we investigated the mono alkylation abilities of FR66979 with respect to a number of inosine-substituted oligonucleotides and observed that the FR900482 class of compounds were able to give rise to easily separable orientation isomers of their respective cross-links. The FR900482 class of antitumor antibiotics cross-link DNA in a fashion analogous to the mitomycins. The cross-linking reaction yields two orientation isomers which are of vastly different electrophoretic mobility and which also exhibit radically different DNA-protein recognition properties upon reaction with AluI restriction endonuclease. In addition, mono-alkylation of DNA by FR66979 shows little, if any, dependence upon pre-covalent interactions deemed necessary for the mitomycins. These insights support the proposal that the FR900482 class of compounds represents a compelling clinical replacement for mitomycin C, given its greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy.

Mutational specificity of the syn 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene.

The mutational specificity of the syn dihydrodiol epoxide of 5-methylchrysene in the supF gene of the pSP189 vector was examined. Transversion mutations at GC pairs predominated with G --> T and G --> C changes accounting for 42 and 21% of total base change mutations. The types of mutations found reflect the previously determined chemical preference of this reactive species for reaction with deoxyguanosine residues in DNA.

Cytotoxicity, DNA binding, and reactivity against nucleosides of platinum (II) and (IV) spermine compounds

We describe the synthesis and characterization of a [sperH 4][PtCl 4] 2 salt and of five binuclear platinum (II) and (IV)-spermine compounds of formula [(PtCl 2) 2(sper)], cis-[(Pt(CH 2(COO) 2)(sper)], and cis-[(PtCl 4) 2(sper)], (CBDCA= 1,1′-cyclobutanedicarboxylate, cis-trans-c0s[(PtCl 2(OH) 2) 2(sper)], and cis-[(PtCl 4) 2(sper)], respectively. The 1H and 195Pt-NMR analysis of the complexes formed between these compounds and nucleosides indicated that the Pt centers show preferential binding to the N(7) of guanosine and adenosine residues, also being capable of forming bridged structures through the N(7) and N(1). The synthesized Pt-spermine compounds do not form complexes with cytidine residues at 37°C. The circular dichroism, melting, and electrophoretic data of the compounds—DNA complexes show that the Pt(IV)-spermine complezes induce lower DNA conformational changes than their Pt(II) analogs. These results correlate with the IC 50 values obtained against MDA-MB 468 and HL-60 human cancer cells which are higher than those of cis-DDP. The [sperH 4][PtCl 4] 2 salt produces a high level of DNA modification and exhibits IC 50 values lower than those of cis-DDP.

Effect of G-rich sequences on the synthesis, purification, hybridization, cell uptake, and hemolytic activity of oligonucleotides

We designed G-rich oligonucleotides in which the position and length of the guanosine residues were modified and studied the effects of these contiguous guanosine residues on the synthesis, purification, hybridization, cell uptake, and hemolytic effect of oligonucleotides. Our results revealed that the hyperstructure formation of these oligonucleotides depended on the length and position of the guanosine residues and the flanking sequences. The phosphorothioate oligonucleotides formed much less hyperstructures than their phosphodiester counterpart. These hyperstructures were more stable towards nucleases, were taken up by cells efficiently, and showed pronounced polyanionic related side-effects. Several phosphodiester and phosphorothioate G-rich oligonucleotides were synthesized and evaluated in respect of their ability to form hyperstructures and their hybridization characteristics. Additionally, their cell uptake and hemolytic activity were studied.

Effect of DNA-binding Drugs on Early Growth Response Factor-1 and TATA Box-binding Protein Complex Formation with the Herpes Simplex Virus Latency Promoter

Adjacent binding sites for early growth response factor-1 (EGR1) and TATA box-binding protein (TBP) were identified on the herpes simplex virus latency promoter in previous work. The binding of EGR1 to the GC-rich region prevented TBP binding to the AT-rich region. With the simultaneous addition of both EGR1 and TBP, the intercalator nogalamycin prevented EGR1 complex formation, resulting in a dose-dependent increase of the TBP.DNA complex. The minor groove binder chromomycin A3 inhibited EGR1 complex formation but resulted in a smaller increase of the TBP complex. In contrast, an alkylating intercalator hedamycin strongly inhibited binding of both proteins. The ability of these GC-binding drugs to prevent EGR1.DNA complex formation was in the following order: hedamycin > nogalamycin > chromomycin A3, and the specificity was nogalamycin > chromomycin A3 > hedamycin. With transcription factor IIA (TFIIA) in the assay, TBP was able to bind the promoter whereas formation of the EGR1.DNA complex was reduced. An AT minor groove-binding drug, distamycin A, disrupted the TBP.TFIIA.DNA complex and restored the EGR1.DNA complex. We conclude that the binding motif and sequence preference of DNA-interactive drugs are manifested in their ability to inhibit the transcription factor-DNA complexes.

Specific guanylation of Lupinus luteus 5S rRNA at its 3' end in HeLa cell extract.

We have found that Lupinus luteus 5S ribosomal RNA is post-transcriptionally modified at its 3' end by addition of pG catalysed by HeLa cells extract. This extra nucleotide is not encoded within Lupinus luteus 5S rRNA gene. A similar reaction has been previously observed for eukaryotic histidine tRNAs. However an additional guanosine residue was ligated post-transcriptionally at the 5' end of that tRNA.

Reaction of guanosine with glucose, ribose, and glucose 6-phosphate

The reaction of guanosine with glucose, glucose 6-phosphate or ribose in the presence of propylamine leads to the formation of several guanosine derivatives. During this process sugar degradation products are attached to the amino group of the guanosine residue. N 2-(1-Carboxy-3,4-dihydroxybutyl)-guanosine, N 2-(1-carboxyethyl)-guanosine, N 2-[1-( N-propyl-carbamoyl)-ethyl]-guanosine, and N 2-[1-( N-propyl-carbamoyl)-methyl]-guanosine have been isolated as pure compounds. N 2-[1-( N-Propyl-carbamoyl)-3,4,5-trihydroxypentyl]-guanosine and N 2-[1-( N-propyl-carbamoyl)-3,4-dihydroxybutyl]-guanosine, which were synthesized, can be detected in glucose- and ribose-guanosine reaction mixtures.

An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the Tetrahymena group I intron.

On the basis of the nucleotide sequence of Tetrahymena group I intron, we constructed a 31 residue RNA that has the P7 stem and the 3'-terminal guanosine residue (3'-G) with a putative stem-loop structure (P9.0) intervening between them. For this model RNA (P7/P9.0/G), four residues around the guanosine binding site (GBS) in the P7 stem were found to exhibit much lower sensitivities to ribonuclease V1 than those of a variant RNA having adenosine in place of the 3'-G, suggesting that the 3'-G contacts around the GBS. NMR analyses of the imino proton resonances of the P7/P9.0/G RNA indicated that the base pairing in the GBS is retained on the interaction with the 3'-G, and that the two base pairs of the putative P9.0 stem-loop are definitely formed. Comparison of the RNA with its variants with either A (3'-A) or a deletion in place of the 3'-G suggested that the stability of the P9.0 stem-loop is affected by the GBS-3'-G interaction. The melting temperatures of the P9.0 stem-loop were determined from the UV absorbances of these RNAs, which quantitatively indicated that the P9.0 stem-loop is significantly stabilized by the interaction of the GBS with the 3'-G, rather than the 3'-A, and also by direct interaction with divalent cations (Mg2+, Ca2+ or Mn2+). Upon replacement of the G-C base pair by C-G in the GBS of the P7/P9.0/G RNA, the specificity was switched from 3'-G to 3'-A, as in the case of the intact intron.

Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage.

Induction of cytochrome P4501A1 (CYP1A1) in the hepatoma Hepa1c1c7 cell line results in an elevation in the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage and the major repair product of 8-oxo-2'-deoxyguanosine (oxo8dG) residues in DNA. Treatment of this cell line with 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), a nonmetabolized environmental contaminant, and indolo(3,2-b)carbazole (ICZ), a metabolite of a natural pesticide found in cruciferous vegetables, is shown to both induce CYP1A1 activity and elevate the excretion rate of oxo8Gua; 7,8-benzoflavone (7,8-BF or alpha-naphthoflavone), an inhibitor of CYP1A1 activity and an antagonist of the aryl hydrocarbon (Ah) receptor, reduced the excretion rate of oxo8Gua. The essential role of Ah-receptor, which mediates the induction of CYP1A1, is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in Ah receptor-defective c4 mutant cells. While there was a significant 7.0-fold increase over 2 days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa1c1c7 cells compared to control, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair. Thus, the induction of CYP1A1 appears to lead to a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer.

Site-specific substitution of inosine at the terminal positions of a pre-mRNA intron: Implications for the configuration of the terminal base interaction

Genetic evidence in yeast has revealed that a non-Watson-Crick base-pairing interaction between terminal guanosine residues of the intron is required for the second step of pre-mRNA splicing. To explore the likely configuration of the interaction between the terminal guanosines of the intron, inosine was uniformly incorporated into an adenovirus pre-mRNA substrate (Ade) to replace guanosine residues. Splicing of the inosine-containing Ade pre-mRNA was completely inhibited. Psoralen cross-linking reveals that the association of U1 and U2 snRNPs with the intron was impaired. To eliminate the deleterious effects caused by complete inosine replacement, guanosine residues at the splice site(s) of the Ade pre-mRNA were substituted by inosine. Such pre-mRNA substrates were obtained by ligation of two or three RNA fragments; the 3′ piece was primed with inosine or mono-phosphate inosine. Splicing of the Ade pre-mRNA containing an inosine residue at the 5′ or the 3′ splice site, or at both sites proceeds normally. Thus, the functions of the terminal guanosine residues of the intron in splicing can be replaced by inosine. This result supports the previous notion that an N1-carbonyl symmetric interaction likely occurs between the intron terminal residues during pre-mRNA splicing.

Iron bound to the lipophilic iron chelator, 8-hydroxyquinoline, causes DNA strand breakage in cultured lung cells.

The formation of DNA-strand breaks was studied in cultured human lung cells (A 549) subjected to iron, either in the form of iron(III) citrate or in combination with the metal chelators ethylene diamine tetra-acetic acid (EDTA), nitrilo triacetic acid (NTA), or 8-hydroxyquinoline (8HQ). After 15 min exposure to 5 microM iron(III) citrate or iron chelate, the cellular levels of iron were found to be three times higher in cells subjected to iron-8HQ than in cells subjected to iron(III) citrate, iron-EDTA or iron-NTA. Exposure to iron-8HQ caused extensive DNA-strand breakage, whereas no such breakage was found in cells exposed to iron-EDTA or iron-NTA. The DNA damage caused by iron-8HQ increased with time and dose, and DNA-strand breakage was clearly demonstrable in cells after 15 min exposure to as little as 0.1 microM iron-8HQ. Moreover, iron-8HQ was strongly toxic to the cells and inhibited their growth after exposure. Along with the formation of DNA-strand breaks, the concentration of cellular malondialdehyde increased four-fold after exposure to iron-8HQ and two-fold after exposure to iron-EDTA or iron-NTA, suggesting that reactive oxygen metabolites might be involved in the toxic action. Moreover, both iron-EDTA and iron-NTA caused a considerable hydroxylation of deoxyguanosine (dG) residues in DNA in vitro, whereas iron(III) citrate and iron-8HQ only caused a minor hydroxylation of dG. This points to the possibility that iron-8HQ-mediated DNA-strand breakage in cells might be due to the action of a metal-bound oxyl radical formed from the iron-8HQ complex rather than to the formation of hydroxyl radicals. Altogether, these findings indicate that iron bound to the lipophilic chelator, 8HQ, has strong toxic properties and that it may cause substantial DNA-strand breakage and lipid peroxidation in living cells.

Interaction of the periplasmic dG-selective Streptomyces antibioticus nuclease with oligodeoxynucleotide substrates.

The interaction of a periplasmic nuclease, isolated from Streptomyces antibioticus, with several oligodeoxynucleotide substrates has been studied. Double-stranded oligonucleotides that contain sequences of four or more consecutive deoxyguanosine residues are preferentially hydrolyzed, with the strongest cutting site occurring at GGG decreases G. The enzyme does not hydrolyze these sequences in single-stranded DNA. However the sequence selectivity of the nuclease is far from absolute. Other sequences can also be cut, albeit more poorly, and differences in cutting rates are observed for runs of dG bases that differ in their flanking sequences. An oligonucleotide, thirty-six bases in length, that contains a central run of five dG bases has been used to evaluate the importance of the individual deoxyguanosines in recognition and cleavage. With this oligonucleotide cutting takes place at GG[symbol: see text]G decreases G[symbol: see text]G (decreases, most prominent cut; [symbol: see text], less prominent cuts). The use of dG base analogues revealed that two bases, one and two steps removed from the cleavage site in the 5' direction (*G*GG decreases), were of most importance in the determination of the nuclease DNA cleavage selectivity. Of these the inner starred dG was the most critical. The use of 5-methyldeoxycytidine also showed that the dC, base paired to this critical dG, influenced cleavage specificity. The overall pattern of results seen with the base analogues suggested that the nuclease interacted with both strands of the DNA and also contacted the nucleic acid in both the major and minor grooves. Gel retardation analysis together with footprinting experiments using hydroxyl radicals, dimethyl sulfate, and ethylnitrosourea indicated that the nuclease does not form a tight and specific complex with sequences containing dG runs, at least in the absence of the essential co-factor, Mg2+.

Solution conformation of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dC in a DNA duplex.

Combined NMR-molecular mechanics computational studies were undertaken on the C8-deoxyguanosine adduct formed by the carcinogen 1-nitropyrene embedded in the d(C5-[AP]G6-C7).d(G16-C17-G18) sequence context in a 11-mer duplex, with dC opposite the modified deoxyguanosine. The exchangeable and nonexchangeable protons of the aminopyrene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of several proton resonances for the three nucleotide d(G16-C17-G18) segment positioned opposite the [AP]dG6 lesion site resulting in weaker NOEs involving these protons in the adduct duplex. The solution conformation of the [AP]dG.dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine ring of [AP]dG6 is displaced into the major groove and stacks with the major groove edge of dC5 in the adduct duplex. Both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove as a consequence of the aminopyrene ring intercalation into the helix. This base-displaced intercalative structure of the [AP]dG.dC 11-mer duplex exhibits several unusually shifted proton resonances which can be accounted for by the ring current contributions of the deoxyguanosinyl and pyrenyl rings of the [AP]dG6 adduct. In summary, intercalation of the aminopyrene moiety is accompanied by displacement of both [AP]dG6 and the partner dC17 into the major groove in the [AP]dG.dC 11-mer duplex.

Variation of gonococcal lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes encoding glycosyl transferases.

The lipooligosaccharide (LOS) expressed by gonococci spontaneously varies its structure at high frequency, but the underlying genetic mechanism has not been described. We have previously reported that the genes encoding the glycosyl transferases responsible for the biosynthesis of the variable alpha chain of the LOS of Neisseria gonorrhoeae are located in a locus containing five genes, lgtA, lgtB, lgtC, lgtD, and lgtE. Sequence analysis showed that lgtA, lgtC, and lgtD contained poly-G tracts within the coding frames, leading to the hypothesis that shifts in the number of guanosine residues in the poly-G tracts might be responsible for the high frequency variation in structure of gonococcal LOS. We now provide experimental evidence confirming this hypothesis.

Conformational switches involved in orchestrating the successive steps of group I RNA splicing.

Group I introns possess a conserved guanosine residue at their 3' end, termed omegaG, that, in the case of the Tetrahymena pre-rRNA, is a major determinant of the second step of splicing. We examined the role of omegaG in self-splicing of the 249-residue group I intron of the Anabaena PCC7120 tRNAleu precursor. Contrary to observations with the Tetrahymena pre-rRNA intron, a mutation that places an adenosine residue at the omega position did not have a severe effect on the second step of splicing; neither 3' splice-site selection nor the rate of the second step was altered. The first step of splicing, however, was now readily reversed. This unexpected effect also resulted from a mutation that altered the nucleoside specificity of the intronic guanosine-binding site. The theme common to these mutations is that reversal of the first step of splicing results when there is not a strong interaction between the guanosine-binding site and the omega residue. This suggests that a major role of omegaG is to compete with the exogenous guanosine molecule added to the intron in the first step of splicing for the single guanosine-binding site of the intron. From these data, we are able to extend the mechanism for the self-splicing reaction of this intron by proposing two distinct conformational changes between the first and second steps of the splicing. The first of these is the exchange of the exogenous nucleoside for the omega nucleoside. This is the equilibrium that we can perturb by mutations at either the omega position or the guanosine-binding site. An additional conformational change then fully activates the intron for the second step of splicing.

Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing.

Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.

Solution conformation of [AF]dG opposite a -2 deletion site in a DNA duplex: intercalation of the covalently attached aminofluorene ring into the helix with base displacement of the C8-modified syn guanine and adjacent unpaired 3'-adenine into the major groove.

This paper reports the solution conformation of the covalent aminofluorene-C8-deoxyguanosine [AF]dG adduct positioned opposite a -2 deletion site in a DNA oligomer duplex. The combined NMR and molecular mechanics computational studies were undertaken on the [AF]dG adduct embedded in the d(C5-[AF]G6-A7-C8).d(G17-G18) sequence context in a duplex containing 12 residues on the modified strand and 10 on the partner strand, with no bases opposite the [AF]dG6-dA7 segment. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the [AF]dG.2del 12-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminofluorene ring of [AF]dG6 is intercalated between intact Waston-Crick dC5.dG18 and dC8.dG17 base pairs with the deoxyguanosine base of [AF]dG6 in a syn alignment displaced into the major groove. The syn glycosidic torsion angle at [AF]dG6 is supported by both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue. The unpaired dA7 base is also looped out of the helix into the major groove with the purine rings of [AF]dG6 and dA7 stacking on each other in the groove. The long axis of the intercalated aminofluorene ring is parallel to the long axis of the flanking dG.dC base pairs. The intercalation site is wedge shaped with a pronounced propeller-twisting and buckling of the dC5.dG18 base pair. The deoxyguanosine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative base displacement structure of the [AF]dG.2del 12-mer duplex exhibits several unusually shifted proton resonances that can be readily accounted for by the ring current contributions of the deoxyguanosine purine and carcinogen fluorene aromatic rings of the [AF]dG6 adduct. We note similarities between the present conformation of [AF]dG positioned opposite a -2 deletion site with our earlier conformational studies of [AF]dG positioned opposite a -1 deletion site [Mao, B., Cosman, M., Hingerty, B. E., Broyde, S., & Patel, D. J. (1995) Biochemistry 34, 6226-6238]. For both conformations, the aminofluorene carcinogen inserts into the helix at the deletion site through base displacement of the modified deoxyguanosine in a syn alignment into the major groove and directed toward its 5'-neighbor in the sequence. These structures provide a molecular explanation of how transient strand slippage of the lesion-containing segment can be accommodated by a double helix following translesion synthesis.

The Structure of a Novel DNA Duplex Formed by Human Centromere d(TGGAA) Repeats with Possible Implications for Chromosome Attachment during Mitosis

The solution structure of the DNA duplex [GT GGAAT GGAAC] 2containing a tandem repeat of the human centromere (TGGAA) nunit has been determined by two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics/energy minimization (MD/EM) methods. This remarkably stable "self-complementary" antipar allel duplex contains a tandem repeated motif in which unpaired guanine residues from opposite strands intercalate and costack between sheared G·A pairs. Twelve independent refined structures were determined from the NMR data and found to converge to a single family of closely related structures with pair-wise r.m.s.d. values of 0.55±0.25 Å. All sugar residues are in the normal C2′- endoconformation except for the unpaired guanosines, which are in the unusual C3′- endoconformation. The guanosine residues of the bracketing G·A pairs have high-antiglycosidic torsion angles and ζ backbone torsion angles close to the transdomain. The structure exhibits many unusual interstrand interactions, including base-sugar stacking, base-phosphate hydrogen bonding and cross-strand base stacking. The [GGA] 2unit contains a stack of four contiguous guanine residues, all of which have their hydrogen-bonding surface (N 2H-N1H-O 6- N7) exposed to solvent and available for interaction with other bases or ligands. This unexpected property may explain the unique morphology and function of the human centromere in mitosis.

Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis.

Lipopolysaccharide (LPS) is a major determinant of Neisseria, meningitidis virulence. A key feature of meningococcal LPS is the phase-variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase-variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae, was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic-resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine-SDS-PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto-N-neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5'-end of the coding sequence. As the lacto-N-neotetraose structure in meningococcal LPS is subject to phase-variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an 'off' phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped-strand mispairing mechanism, operating in the first ORF, controls the phase-variable expression of lacto-N-neotetraose.

The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast.

The m6(2)A1779m6(2)A1780 dimethylation at the 3' end of the small subunit rRNA has been conserved in evolution from bacteria to eukaryotes. The yeast 18S rRNA dimethylase gene DIM1 was cloned previously by complementation in Escherichia coli and shown to be essential for viability in yeast. A conditional GAL10::dim1 strain was constructed to allow the depletion of Dim1p from the cell. During depletion, dimethylation of the pre-rRNA is progressively inhibited and pre-rRNA processing at cleavage sites A1 and A2 is concomitantly lost. In consequence, the mature 18S rRNA and its 20S precursor drastically underaccumulate. This has the effect of preventing the synthesis of nonmethylated rRNA. To test whether the processing defect is a consequence of the absence of the dimethylated nucleotides or of the Dim1p dimethylase itself, a cis-acting mutation was created in which both dimethylated adenosines are replaced by guanosine residues. Methylation cannot occur on this mutant pre-rRNA, but no clear pre-rRNA processing defect is seen. Moreover, methylation of the wild-type pre-rRNA predominantly occurs after cleavage at sites A1 and A2. This shows that formation of the m6(2)A1779m6(2)A1780 dimethylation is not required for pre-rRNA processing. We propose that the binding of Dim1p to the pre-ribosomal particle is monitored to ensure that only dimethylated pre-rRNA molecules are processed to 18S rRNA.