Regulation by nutrition and age of insulin-like growth factor binding sites in ovine kidney

A combined NMR-molecular dynamics approach has been applied to determine the solution structure of a truncated analogue of the Bombyx mori telomeric d(TTAGG) single repeat sequence in Na+ cation-containing aqueous solution. The two-fold symmetric four-stranded d(TAGG) quadruplex contains two adjacent G(syn).G(syn).G(anti).G(anti) G-tetrads sandwiched between novel (T.A).A triads with individual strands having both a parallel and antiparallel neighbour around the quadruplex. The (T.A).A triad represents the first experimental verification of a base triad alignment which constitutes a key postulate in the recently proposed model of triad-DNA. Further, the (T.A).A triad is generated by positioning an A residue through hydrogen bonding in the minor groove of a Watson-Crick T.A base pair and includes a T-A platform related to an A-A platform recently observed in the structure of the P4-P6 domain of the Tetrahymena self splicing group I ribozyme. The novel architecture of the truncated Bombyx mori quadruplex structure sets the stage for the design and potential identification of additional base tetrads and triads that could participate in pairing alignments of multi-stranded DNA structures during chromosome association and genetic recombination.     

Solution structure of the d(T-C-G-A) duplex at acidic pH. A parallel-stranded helix containing C+ .C, G.G and A.A pairs

The solution structure of the d(T-C-G-A) sequence at acidic pH has been determined by a combination of NMR and molecular dynamics calculations including NOE intensity based refinements. This sequence forms a right-handed parallel-stranded duplex with C+ .C (three hydrogen bonds along Watson-Crick edge), G.G (two symmetry related N2-H.. N3 hydrogen bonds) and A.A (two symmetry related N6-H..N7 hydrogen bonds) homo base-pair formation at acidic pH. The duplex is stabilized by intra-strand base stacking at the C2-G3 step and cross-strand base stacking at the G3-A4 step. The thymine residues on partner strands are directed towards each other and are positioned over the C+ .C base-pair. All four residues adopt anti glycosidic torsion angles and C2'-endo type sugar conformations in the parallel-stranded d(T-C-G-A) duplex which exhibits large changes in twist angles between adjacent steps along the duplex. This study rules out previously proposed models for the structure of the d(T-C-G-A) duplex at acidic pH and supports earlier structural contributions, which established that d(C-G) and d(C-G-A) containing sequences at acidic pH pair through parallel-stranded alignment. We have also monitored hydration patterns in the symmetry related grooves of the parallel-stranded d(T-C-G-A) duplex.     

Cytosine-cytosine+ base pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect

Previous spectroscopic studies demonstrated that the oligodeoxynucleotide d(CGC G3 GCG) undergoes a reversible cation-dependent transition between Watson-Crick (WC) hairpin and parallel-stranded "G-DNA" quadruplex structures [Hardin, C.C., Watson, T., Corregan, M., & Bailey, C. (1992) Biochemistry 31, 833-841]. The relative stabilities of the structures were assessed as a function of pH, and it was found that the quadruplex was substantially stabilized (delta Tm = +15 degrees C) when the pH was shifted from 7.5 to 6 (apparent pKa = 6.8). In the present study, the effects of different cations and pH on four specific sequence varients were determined to test the proposal that this stabilization is due to C.C+ base pair formation mediated by N3-protonation of cytosine. Characteristically large differences in stability were observed when structures formed by d(TAT G3 ATA) and d(TAT G4 ATA) were thermally dissociated at pH 7 in the presence of different cations, verifying that Gn tracts bordered by TAT- and -ATA sequences form quadruplex structures. Imino proton NMR results indicate that the d(m5C G m5C G3 G m5C G)4 and d(TAT G4 ATA)4 quadruplex structures are parallel-stranded. It was necessary to increase the K+ concentration from 40 mM to ca. 200 mM to stabilize d(TAT G3 ATA)4, while the d(TAT G4 ATA)4 complex was nearly as stable as the quadruplex formed by d(CGC G3 GCG) under the same conditions. The d(TAT G4 ATA)4 quadruplex was only slightly stabilized at pH 6 relative to pH 7.5 (delta Tm = +3 degrees C), confirming that the unique stabilization that occurs in the pH 6.8 range with [d(CGC Gn GCG)4.ionn] complexes is due to the C residues. The sequence d(m5C G m5C G3 G m5C G) was found to form a very stable quadruplex in K+ or Ca2+. As with the quadruplex formed by the unmethylated analog, the stability is greatly enhanced when the pH is decreased below about 7.2 (pKa,obs = 6.8). Dissociation kinetic constants and activation energies were determined for quadruplexes formed by d(CGC G3 GCG), d(m5C G m5C G3 G m5C G) and d(TAT G4 ATA). Quantitative comparisons showed that methylation produces a complex that is much more stable at pH 7 in 40 mM Na+ than either of the unmodified structures; the rate-limiting activation energy for dissociation of d(CGC G3 GCG)4 was 22 kcal mol-1 less than for the methylated analog.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural basis of DNA recognition and mechanism of quadruplex formation by the beta subunit of the Oxytricha telomere binding protein

Interactions of the beta subunit of the Oxytricha nova telomere binding protein with the telomeric DNA sequences, d(T4G4)2 and dT6(T4G4)2, have been investigated in vitro using Raman and fluorescence spectroscopies. Raman difference spectra show that the beta subunit binds to both d(T4G4)2 and dT6(T4G4)2 but promotes the formation of a parallel-stranded quadruplex only in dT6(T4G4)2, thus demonstrating the importance of the telomeric 5' tail for in vitro recognition and guanine quadruplex formation. While d(T4G4)2 is not a suitable substrate for quadruplex promotion by the beta subunit, the Raman spectra reveal other structural rearrangements of this DNA strand upon beta subunit binding, including changes in guanine glycosyl torsion angles from syn to anti and disruption of carbonyl hydrogen-bonding interactions. The conformation of d(T4G4)2 in the beta:d(T4G4)2 complex is suggested as a plausible intermediate along the pathway to formation of the parallel-stranded guanine quadruplex. Fluorescence band shifts indicate that at least one of the two tryptophans of the beta subunit is shielded from solvent as a consequence of DNA binding in both the beta:dT6(T4G4)2 and beta:d(T4G4)2 complexes. However, the Raman spectra of these complexes suggest no significant changes in the beta subunit secondary structure attendant with DNA binding. A model for beta subunit binding by Oxytricha telomeric DNA sequences and a mechanism for quadruplex formation are proposed. A key feature of this model is the use of a telomeric hairpin secondary structure as the recognition motif.     

Effect of thymine tract length on the structure and stability of model telomeric sequences

DNA from the telomeres at the ends of eukaryotic chromosomes contains a stretch of simple tandemly repeated sequences in which clusters of G residues alternate with clusters of T/A sequences along one DNA strand. Model telomeric G-clusters form four-stranded structures in Na+ or K+, stabilized by Hoogsteen pairing between G bases. DNA containing a single copy of the G-cluster can self-associate to form tetramers, with a parallel-stranded, right-handed helical structure. Two copies of the 3'-terminal G strand form a folded-back hairpin that dimerizes to create an antiparallel quadruplex structure. We show here that the tetrameric structure is strongly influenced by the T residue flanking either side of the G-cluster. The parallel tetraplex formed by single copies of the sequences dTnG4 is most stable for n = 1 and least stable for n = 8, the longest tract we have studied. At least two thymine residues are required to allow formation of antiparallel folded-back hairpin dimers from two-copy oligomers of sequence d(TnG4)2 in Na+; additional T's destabilize this structure. In K+, the predominant structure formed is the four-stranded parallel tetramer in all cases. Kinetic analysis indicates that the quadruplex structure formed by Oxytricha telomeric DNA overhangs in the presence of Na+ arises by dimerization of two Hoogsteen base-paired hairpins, with a relatively low energy barrier.     

Isolation and characterization of a monoclonal anti-quadruplex DNA antibody from autoimmune "viable motheaten" mice

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library derived from 3-month-old nonimmunized autoimmune, immunodeficient "viable motheaten" mice. This antibody has been tested extensively in vitro and found to bind specifically to DNA quadruplex structures formed by two biologically relevant sequence motifs. Scatchard and nonlinear regression analyses using both one- and two-site models were used to derive association constants for the antibody-DNA binding reactions. In both cases, quadruplexes had higher association constants than triplex and duplex molecules. The anti-quadruplex antibody binds to the quadruplex formed by the promoter-region-derived oligonucleotide d(CGCG4GCG) (Ka = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-derived quadruplexes formed by the oligonucleotides d(TG4) and d(T2G4T2G4T2G4T2G4) (Ka = 5.38 x 10(6) and 1.66 x 10(7) M-1, respectively). The antibody binds both types of quadruplexes but has preferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti-DNA quadruplex antibodies from anti-quadruplex antibody-producing tissue culture supernatants have at least 10-fold higher affinity for quadruplexes than for triplex and duplex DNA structures of similar base composition and length. The antibody binds intramolecular DNA triplexes formed by d(G4T3G4T3C4) and d(C4T3G4T3G4), and the duplex d(CGCGCGCGCG)2 with an affinities of 6. 76 x 10(5), 5.59 x 10(5), and 8.26 x 10(5) M-1, respectively. Competition experiments showed that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadruplex is bound structure-specifically. To our knowledge, this is the first immunological reagent known to specifically recognize quadruplex structures. Subsequent sequence analysis demonstrates homologies between the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to control cell aging via telomeric DNA interactions. The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.     

Conformations of DNA duplexes containing 8-oxoguanine

As a step towards elucidating the mechanisms of mutagenesis induced by irradiation and oxidation, we study the incorporation of 8-oxoguanine (OG) into duplex DNA. Molecular modelling is used to reveal changes in DNA conformational parameters due to mispairs within the sequences d(A5XA5).d(T5YT5) and d(G5XG5).d(C5YC5) where one of the bases of the bases of the central X:Y pair is OG and the other A,T,G or C. The G:C to OG:C replacements in DNA duplexes produce only minor conformational changes, similar to normal base sequence effects. The calculations suggest that both OG(syn):G and OG(syn):A mispairs can also be introduced without drastic distortion of sugar-phosphate backbone. The distortions produced by OG-containing mispairs are also found to be sequence dependent. Overall these calculations suggest that the G-->OG conversion could be an important factor in the irradiative or oxidative damage of DNA.     

Nucleotide sequence of bacteriophage G4 DNA

The 5,577 nucleotide long sequence of bacteriophage G4 DNA has been determined using the 'plus and minus' and chain termination methods of DNA sequencing. This sequence has been compared with that of the closely related bacteriophage phiX174 (refs 1, 55). In the coding regions there is an average of 33.1% nucleotide sequence differences between the two genomes, but the distribution of these changes is not random and the sequence of some genes is more conserved than others. There is less sequence similarity between the untranslated intergenic regions of G4 and phiX174, but despite this the sequences of the J/F, F/G and H/A untranslated spaces in both genomes have similar sized hairpin loops, which may be related to their function.     

Direct binding of p130(Cas) to the guanine nucleotide exchange factor C3G

p130(Cas) (Cas; crk-associated substrate) belongs to a new family of docking molecules. It contains one Src homology (SH) 3 domain in its amino-terminal region followed by a region containing binding motifs for SH2 and SH3 domains. To gain further insight into Cas signaling we used the SH3 domain of Cas in a two-hybrid screen to search a human placenta library for binding partners. The screen confirmed a previous finding of its binding to the focal adhesion kinase (FAK) but also identified C3G, a guanine nucleotide exchange factor. We found direct interaction between Cas and C3G in vitro and in vivo. A series of analysis with C3G deletion mutants revealed a proline-rich Cas-binding site (Ala0-Pro1-Pro2-Lys3-Pro4-Pro5-Leu6-Pro7) located NH2-terminal to the previously characterized Crk binding motifs in C3G. Mutagenesis studies showed that Pro1, Lys3, and Pro4 within the ligand-binding site are critical for high affinity interaction. These results, combined with sequence alignments of proline-rich binding elements from proteins known for Cas binding, define the consensus sequence XXPXKPX which is recognized by the CasSH3 domain. Cas shows structural characteristics of a docking molecule and may serve to bring C3G to specific compartments within the cell.     

Structural alignment of the (+)-trans-anti-benzo[a]pyrene-dG adduct positioned opposite dC at a DNA template-primer junction

This study reports on the solution conformation of the covalent (+)-trans-anti-[BP]dG adduct (derived from the binding of the highly mutagenic and tumorigenic (+)-anti-benzo[a]pyrene diol epoxide to the N2 of deoxyguanosine) positioned opposite dC at a junctional site in the d(A1-A2-C3-[BP]G4-C5- T6-A7-C8-C9-A10-T11-C12-C13).d(G14-G15-A16-T17-+ ++G18-G19-T20-A21-G22-C23) 13/10-mer DNA sequence. The 13-mer represents the template strand containing the junction [BP]dG4 lesion while the complementary 10-mer models a primer strand which extends upto and is complementary to the modified dG4 residue. The solution conformation has been determined by initially incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space and subsequently through restrained molecular dynamics calculations based on a NOE distance and intensity refinement protocol. The duplex segment retains a minimally perturbed B-DNA conformation with all base pairs, including the junctional [BP]dG4.dC23 pair, in Watson-Crick hydrogen-bonded alignments. The pyrenyl ring is not stacked over the adjacent dC5.dG22 base pair but is positioned on the minor groove-side of the [BP]dG moiety and directed toward the 5'-end of the template strand. The pyrenyl ring stacks over the base of the non-adjacent dA2 residue in one direction and the sugar ring of dC23 in the other direction. The solution structure of the (+)-trans-anti-[BP]dG adduct opposite dC in the 13/10-mer in which the modified deoxyguanosine adopts an anti glycosidic torsion angle (this study) is in striking contrast to the structure of the same (+)-trans-anti-[BP]dG moiety in a 13/9-mer of the same sequence but without the dC23 residue positioned opposite the adduct site [Cosman, M., et al. (1995) Biochemistry 34, 15334-15350]. For the latter case, the aromatic portion of the BP residue stacks over the adjacent dC5.dG22 base pair, the modified deoxyguanosine adopts a syn glycosidic torsion angle and is displaced toward the major groove direction. Insights into the factors that affect the sequence and context dependent conformations of stereoisomeric [BP]dG lesions have emerged following comparison of these two structures with the minor groove conformations of the same (+)-trans-anti-[BP]dG lesion in the fully complementary 11-mer duplex [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918] and in the base displaced-intercalative conformation of the 11/10-mer deletion duplex containing a -1 deletion site opposite the lesion [Cosman, M., et al. (1994) Biochemistry 33, 11507-11517]. The contributing factors where applicable include Watson-Crick base pairing at the site of the lesion, positioning of the carcinogen within the floor of the minor groove, and the tendency of the bulky hydrophobic aromatic BP residue to assume stacked or intercalative conformations.     

Molecular cloning of human G alpha q cDNA and chromosomal localization of the G alpha q gene (GNAQ) and a processed pseudogene

G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.     

Characteristics of insulin receptor binding to various rat tissues

The solution secondary structure of the Oxytricha nova telomeric 3' overhang, d(T4G4)2, has been investigated by Raman spectroscopy, hydrogen-deuterium exchange kinetics and gel electrophoresis. The electrophoretic mobility of d(T4G4)2 in non-denaturing gels indicates a highly compact conformation, consistent with a hairpin secondary structure. Raman markers show that the d(T4G4)2 hairpin contains equal numbers of C2'-endo/syn and C2'-endo/anti deoxyguanosine conformers, as well as G.G base-pairs of the Hoogsteen type. The hydrogen-deuterium exchange kinetics of d(T4G4)2, monitored by time-resolved Raman spectroscopy, reveal two kinetically distinct classes of guanine imino (N1H) protons. The more slowly exchanging fraction (kN1H(1)=4.6x10(-3) min-1), which represents 50% of N1H groups, is attributed to Hoogsteen-paired residues. The more rapidly exchanging fraction (kN1H(2)>/=0.3 min-1) is attributable to solvent-exposed residues. Raman dynamic probe of the kinetics of guanine C8H-->C8(2)H exchange in d(T4G4)2 reveals modest retardation vis-à-vis dGMP, which rules out quadruplex formation by the telomeric repeat and confirms an ordered secondary structure consistent with a Hoogsteen-paired hairpin. Similar Raman, hydrogen-isotope exchange and electrophoretic mobility experiments on the related telomeric model, dT6(T4G4)2, also reveal a hairpin stabilized by Hoogsteen G.G pairs. Presence of the 5' thymidine tail preceding the Oxytricha telomeric repeat has no apparent effect on the hairpin secondary structure. We propose a molecular model for the hairpin conformation of the Oxytricha nova telomeric repeat and consider its possible roles in mechanisms of telomeric DNA interaction in vitro and telomere function in vivo.     

Intra- and interloop interactions in the folded G quartet structure of Oxytricha telomeric sequence

The antiparallel intramolecular G quartet structure for the 3.5 copy Oxytricha telomeric sequence d(G4T4)3G4 has been established using a combination of spectroscopic and chemical probing methods. In the presence of Na+ ions, this sequence exhibits a circular dichroism spectrum with a positive band at 295 nm and a negative band around 265 nm, characteristic of an antiparallel G quartet structure. Further, we show that d(G4T4)3G4 adopts an antiparallel intramolecular G quartet structure even in K+ unlike d(G4T4G4). KMnO4 probing experiments indicated the existence of intra and interloop interactions in the Na+ induced structure. We have found that K+ not only increases the thermal stability of G quartet structure but also binds to the loop region and disrupts stacking and interloop interactions. Biological consequences of such cation-dependent conformational micro-heterogeneity in the loop region of G quartet structures is also discussed.     

Divalent transition metal cations counteract potassium-induced quadruplex assembly of oligo(dG) sequences

Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.     

A novel parvovirus isolated from Manchurian chipmunks

A novel parvovirus was identified in Manchurian chipmunks inhabiting Korea. Hepatitis B surface antigen (HBsAg) was detected in sera from 4 animals among 62 apparently healthy chipmunks. Electron microscopic examination of the HBsAg-positive sera revealed virus-like spherical particles 20-22 nm in diameter. Extraction of nucleic acid under annealing conditions from the serum samples containing virus-like particles yielded a single species of DNA molecule with the electrophoretic mobility of 5.6-kb double-stranded DNA. Four overlapping clones that encompassed almost the full-length viral genome, except both ends, were obtained. By sequencing these clones, we determined the sequence of 5097 nucleotides of the viral DNA. Two open reading frames were identified, with the left side open reading frame encoding a putative nonstructural protein and the right side open reading frame encoding a putative capsid protein. The nucleotide and amino acid sequences showed significant homology to parvovirus B19 and simian parvovirus, but showed little homology to other mammalian autonomous parvoviruses or adeno-associated viruses. These observations indicate that the virus isolated from Manchurian chipmunks is a novel parvovirus and may be a potentially useful animal model of human B19 infection as a new member of the Erythrovirus genus of the Parvoviridae.     

Directed integration of minute virus of mice DNA into episomes

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.     

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

BACKGROUND: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP) with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution. RESULTS: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G.A mismatches are flanked by sheared G.A and reversed Hoogsteen G.G mismatch pairs. CONCLUSIONS: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G.A mismatch formation. The recognition G.A mismatch stacks with a reversed Hoogsteen G.G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10(14) molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.     

M proteins of group G streptococci: mechanisms of resistance to phagocytosis

Group G streptococci that express M protein and resist phagocytosis in human blood (virulent strains) were compared with strains of groups G and A that are readily phagocytosed (avirulent). Virulent group G streptococci were less effective (P < .05) as activators of the alternative complement pathway (ACP) than were avirulent streptococci. In immunofluorescence studies, C3 bound more avidly to avirulent than to virulent group G streptococci. Resistance of virulent group G strains to ACP opsonization and to phagocytosis was markedly diminished by removal with pepsin of the type-specific portion of the M molecule. Preincubation with fibrinogen did not diminish ACP activation or C3 binding by virulent group G and A streptococci but did exert an antiphagocytic effect. Given the similarity of M proteins of groups G and A in structure and function, other microbial constituents are likely responsible for differences in the spectra of illnesses attributable to the two serogroups.