Showing papers on "Base pair published in 2001"
TL;DR: In this paper, a method for identifying a set of genes where cell cycle regulator binding correlates with gene expression and identifying genomic targets of cell cycle transcription activators in living cells is also encompassed.
Abstract: The present invention relates to a method of identifying a region (one or more) of a genome of a cell to which a protein of interest binds. In the methods described herein, DNA binding protein of a cell is linked (e.g., covalently crosslinked) to genomic DNA of a cell. The genomic DNA to which the DNA binding protein is linked is removed and combined or contacted with DNA comprising a sequence complementary to genomic DNA of the cell under conditions in which hybridization between the identified genomic DNA and the sequence complementary to genomic DNA occurs. Region(s) of hybridization are region(s) of the genome of the cell to which the protein of binds. A method of identifying a set of genes where cell cycle regulator binding correlates with gene expression and of identifying genomic targets of cell cycle transcription activators in living cells is also encompassed.
1,931 citations
TL;DR: The Ku heterodimer (Ku70 and Ku80 subunits) contributes to genomic integrity through its ability to bind DNA double-strand breaks and facilitate repair by the non-homologous end-joining pathway.
Abstract: The Ku heterodimer (Ku70 and Ku80 subunits) contributes to genomic integrity through its ability to bind DNA double-strand breaks and facilitate repair by the non-homologous end-joining pathway. The crystal structure of the human Ku heterodimer was determined both alone and bound to a 55-nucleotide DNA element at 2.7 and 2.5 A resolution, respectively. Ku70 and Ku80 share a common topology and form a dyad-symmetrical molecule with a preformed ring that encircles duplex DNA. The binding site can cradle two full turns of DNA while encircling only the central 3-4 base pairs (bp). Ku makes no contacts with DNA bases and few with the sugar-phosphate backbone, but it fits sterically to major and minor groove contours so as to position the DNA helix in a defined path through the protein ring. These features seem well designed to structurally support broken DNA ends and to bring the DNA helix into phase across the junction during end processing and ligation.
1,188 citations
TL;DR: A simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques is described, which removes the need for lengthy growth periods and traditional DNA isolation methods.
Abstract: We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and phi29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.
1,107 citations
TL;DR: This work proposes a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson-Crick edge, the Hoogsteen edge, and the Sugar edge, which facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences.
Abstract: Non-Watson‐Crick base pairs mediate specific interactions responsible for RNA‐RNA self-assembly and RNA‐ protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson‐Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 29-OH and which has also been referred to as the Shallowgroove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson‐Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.
967 citations
TL;DR: Biosensor elements that are capable of identifying individual DNA strands with single-base resolution are described, exemplified by the detection of a drug resistance–conferring mutation in the reverse transcriptase gene of HIV.
Abstract: We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the alpha-hemolysin (alphaHL) pore to form a "DNA-nanopore". The binding of single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes in the ionic current flowing through a nanopore. On the basis of DNA duplex lifetimes, the DNA-nanopores are able to discriminate between individual DNA strands up to 30 nucleotides in length differing by a single base substitution. This was exemplified by the detection of a drug resistance-conferring mutation in the reverse transcriptase gene of HIV. In addition, the approach was used to sequence a complete codon in an individual DNA strand tethered to a nanopore.
805 citations
TL;DR: Although there is no simple, general code for zinc finger-DNA recognition, selection strategies have been developed that allow these proteins to be targeted to almost any desired site on double-stranded DNA.
Abstract: ▪ Abstract Cys2His2 zinc finger proteins offer a stable and versatile framework for the design of proteins that recognize desired target sites on double-stranded DNA. Individual fingers from these proteins have a simple ββα structure that folds around a central zinc ion, and tandem sets of fingers can contact neighboring subsites of 3–4 base pairs along the major groove of the DNA. Although there is no simple, general code for zinc finger–DNA recognition, selection strategies have been developed that allow these proteins to be targeted to almost any desired site on double-stranded DNA. The affinity and specificity of these new proteins can also be improved by linking more fingers together or by designing proteins that bind as dimers and thus recognize an extended site. These new proteins can then be modified by adding other domains—for activation or repression of transcription, for DNA cleavage, or for other activities. Such designer transcription factors and other new proteins will have important applica...
752 citations
TL;DR: A derived consensus secondary structure for the K‐turn includes 10 consensus nucleotides out of 15, and predicts its presence in the 5′‐UTR of L10 mRNA, helix 78 in Escherichia coli 23S rRNA and human RNase MRP.
Abstract: Analysis of the Haloarcula marismortui large ribosomal subunit has revealed a common RNA structure that we call the kink-turn, or K-turn. The six K-turns in H.marismortui 23S rRNA superimpose with an r.m.s.d. of 1.7 A. There are two K-turns in the structure of Thermus thermophilus 16S rRNA, and the structures of U4 snRNA and L30e mRNA fragments form K-turns. The structure has a kink in the phosphodiester backbone that causes a sharp turn in the RNA helix. Its asymmetric internal loop is flanked by C-G base pairs on one side and sheared G-A base pairs on the other, with an A-minor interaction between these two helical stems. A derived consensus secondary structure for the K-turn includes 10 consensus nucleotides out of 15, and predicts its presence in the 5'-UTR of L10 mRNA, helix 78 in Escherichia coli 23S rRNA and human RNase MRP. Five K-turns in 23S rRNA interact with nine proteins. While the observed K-turns interact with proteins of unrelated structures in different ways, they interact with L7Ae and two homologous proteins in the same way.
742 citations
TL;DR: Crystal structures of Dpo4 in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, have been solved at 1.7 A and 2.1 A resolution, respectively, suggesting a possible mechanism for bypassing thymine dimers.
633 citations
TL;DR: It is shown that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C-->T and G-->A substitutions, which are due to the occurrence of modified deoxycytidine residues in the template DNA.
Abstract: We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C→T and G→A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, ‘jumping PCR’, i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than ∼0.1% even under the unlikely scenario where each amplification starts from a single template molecule.
622 citations
TL;DR: Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.
Abstract: Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.
496 citations
TL;DR: This nanopore detector with a support vector machine (SVM) to analyze DNA hairpin molecules on the millisecond time scale and can discriminate between individual DNA hairpins that differ by one base pair or by one nucleotide.
Abstract: RNA and DNA strands produce ionic current signatures when driven through an α-hemolysin channel by an applied voltage. Here we combine this nanopore detector with a support vector machine (SVM) to analyze DNA hairpin molecules on the millisecond time scale. Measurable properties include duplex stem length, base pair mismatches, and loop length. This nanopore instrument can discriminate between individual DNA hairpins that differ by one base pair or by one nucleotide.
TL;DR: Recent studies have shed new light on the question of what physical forces cause a polymerase enzyme to insert a nucleotide into a strand of DNA and to choose the correct nucleotide over the incorrect ones.
Abstract: ■ Abstract Understanding the mechanisms by which genetic information is replicated is important both to basic knowledge of biological organisms and to many useful applications in biomedical research and biotechnology. One of the main functions of a DNA polymerase enzyme is to help DNA recognize itself with high specificity when a strand is being copied. Recent studies have shed new light on the question of what physical forces cause a polymerase enzyme to insert a nucleotide into a strand of DNA and to choose the correct nucleotide over the incorrect ones. This is discussed in the light of three main forces that govern DNA recognition: base stacking, Watson-Crick hydrogen bonding, and steric interactions. These factors are studied with natural and structurally altered DNA nucleosides.
TL;DR: The data indicate that polymerase η is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.
Abstract: To determine whether DNA polymerase plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.
TL;DR: This review summarizes results concerning molecular interactions of nucleic acid bases as revealed by advanced ab initio quantum chemical (QM) calculations published in last few years and provides an overview of key electronic properties of standard and selected modified nucleobases.
Abstract: This review summarizes results concerning molecular interactions of nucleic acid bases as revealed by advanced ab initio quantum chemical (QM) calculations published in last few years. We first explain advantages and limitations of modern QM calculations of nucleobases and provide a brief history of this still rather new field. Then we provide an overview of key electronic properties of standard and selected modified nucleobases, such as their charge distributions, dipole moments, polarizabilities, proton affinities, tautomeric equilibria, and amino group hybridization. Then we continue with hydrogen bonding of nucleobases, by analyzing energetics of standard base pairs, mismatched base pairs, thio-base pairs, and others. After this, the nature of aromatic stacking interactions is explained. Also, nonclassical interactions in nucleic acids such as interstrand bifurcated hydrogen bonds, interstrand close amino group contacts, C—H … O interbase contacts, sugar–base stacking, intrinsically nonplanar base pairs, out-of-plane hydrogen bonds, and amino–acceptor interactions are commented on. Finally, we overview recent calculations on interactions between nucleic acid bases and metal cations. These studies deal with effects of cation binding on the strength of base pairs, analysis of specific differences among cations, such as the difference between zinc and magnesium, the influence of metalation on protonation and tautomeric equlibria of bases, and cation–π interactions involving nucleobases. In this review, we do not provide methodological details, as these can be found in our preceding reviews. The interrelation between advanced QM approaches and classical molecular dynamics simulations is briefly discussed. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 3–31, 2002; DOI 10.1002/bip.10048
TL;DR: The 3.0 Å resolution structure of wild‐type HIV‐1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine‐rich segment from the HIV‐ 1 genome called the polypurine tract (PPT) is determined.
Abstract: We have determined the 3.0 Å resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The ‘RNase H primer grip’, consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual ‘unzipping’ of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.
TL;DR: Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.
Abstract: A detailed computational analysis of 32 protein-RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein-double-stranded DNA and protein-single-stranded DNA complexes. The interface properties of the protein-RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein-RNA and protein-DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein-RNA complexes, backbone contacts were more dominant in the protein-DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.
TL;DR: In this article, two complexes of [Co(phen)2IP]3+ (IP=imidazo[4,5-f][1,10]phenanthroline) and [Co-(phen) 2PIP] 3+ (PIP=2-phenylimidazo [4, 5-f], 1,10]-phenanthropyline) have been synthesized and characterized by UV/VIS, IR, EA and mass spectra.
TL;DR: The nucleosome core particle is the fundamental repeating subunit of chromatin, and each core particle plus its linker DNA is associated with one molecule of a fifth ‘linker’ histone protein, H1.
Abstract: 1. Introduction 270 1.1 Overview of nucleosome structure 271 2. Relative equilibrium stability (affinity) of histone–DNA interactions in nucleosomes 272 2.1 Relative affinity equals relative equilibrium stability 272 2.2 Competition assays for relative free-energy measurements 273 2.3 Technical issues in relative free-energy measurements 275 2.4 Range of affinities 278 3. Relation of nucleosome stability to nucleosome positioning 279 3.1 Translational nucleosome positioning 279 3.2 Rotational positioning 280 3.3 Unfavorable positioning 281 3.4 Experiments 281 4. Physical basis of DNA sequence preferences 282 4.1 Free-energy cost of DNA bending 283 4.2 Molecular mechanics of DNA bending and bendability 284 4.3 Bent and bendable DNA sequences 286 4.4 Parameter sets for prediction of DNA bending and bendability 288 4.5 DNA twisting 290 4.6 Energetics of nucleosomal DNA packaging 291 5. DNA sequence motifs for nucleosome packaging 292 5.1 Natural and designed nucleosomal DNAs 293 5.2 New rules and reagents from physical selection studies 294 5.3 Molecular basis of DNA sequence preferences 299 5.4 Special properties of the TA step 300 5.5 Unfavorable sequences 302 5.6 Natural genomes 303 5.7 Evolutionary approach toward an optimal sequence 305 5.8 Optimization by design 305 6. Dynamic nucleosome instability 308 6.1 Site-exposure equilibria 308 6.2 DNA sequence-dependence to site-exposure equilibria 312 6.3 Nucleosome translocation 315 6.4 Action of processive enzymes 319 7. Conclusions 319 8. Acknowledgements 320 9. References 320 The nucleosome core particle is the fundamental repeating subunit of chromatin. It consists of two molecules each of the four ‘core histone’ proteins, H2A, H2B, H3 and H4, and a 147 bp stretch of DNA. The lowest level of chromatin organization consists of a repeated array of nucleosome core particles separated by variable lengths of ‘linker DNA’. In many, but not all, cases, each core particle plus its linker DNA is associated with one molecule of a fifth ‘linker’ histone protein, H1. The complex of the core particle plus its linker DNA and H1 (when present) is called a ‘nucleosome’.
TL;DR: The results show that the base-pairing between 5′ and 3′ complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replicate of the tick-borne Flavivirusspecies.
Abstract: A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.
TL;DR: The crystal structure of the catalytic core of S. cerevisiae DNA polymerase eta, determined at 2.25A resolution, reveals a polydactyl right hand-shaped molecule with a unique polymerase-associated domain this article.
TL;DR: It is shown that unwinding is both continuous and processive, occurring at a maximum rate of 972 ± 172 base pairs per second, with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule.
Abstract: RecBCD enzyme is a processive DNA helicase1 and nuclease2 that participates in the repair of chromosomal DNA through homologous recombination3,4. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy5,6. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme7. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 ± 172 base pairs per second (0.30 µm s-1), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.
TL;DR: The tremendous increase in structural information on protein-DNA complexes has uncovered a remarkable structural diversity in DNA binding folds, while at the same time revealing common themes in binding to target sites in the genome.
TL;DR: Analysis of base-substitution hots spots in DNA polymerase error spectra showed that 33 of 36 hotspots in the human polymerase η spectrum conformed to the WA consensus, which suggests errors introduced by this enzyme during synthesis of the nontranscribed DNA strand in variable regions may contribute to strand-specific somatic hypermutagenesis of immunoglobulin genes at A-T base pairs.
Abstract: Mutational spectra analysis of 15 immunoglobulin genes suggested that consensus motifs RGYW and WA were universal descriptors of somatic hypermutation. Highly mutable sites, "hotspots", that matched WA were preferentially found in one DNA strand and RGYW hotspots were found in both strands. Analysis of base-substitution hotspots in DNA polymerase error spectra showed that 33 of 36 hotspots in the human polymerase eta spectrum conformed to the WA consensus. This and four other characteristics of polymerase eta substitution specificity suggest that errors introduced by this enzyme during synthesis of the nontranscribed DNA strand in variable regions may contribute to strand-specific somatic hypermutagenesis of immunoglobulin genes at A-T base pairs.
TL;DR: HMG1 and 2 appear to play important architectural roles in the assembly of nucleoprotein complexes in a variety of biological processes, for example V(D)J recombination, the initiation of transcription, and DNA repair.
Abstract: HMG1 and 2 (high mobility group proteins 1 and 2; renamed HMGB1 and 2) contain two DNA-binding HMG-box domains (A and B) and a long acidic C-terminal domain. They bind DNA without sequence specificity, but have a high affinity for bent or distorted DNA, and bend linear DNA. The individual A and B boxes (which, although broadly similar, show both structural and functional differences) exhibit many of the structure-specific properties of the whole protein. The acidic tail modulates the affinity of the tandem HMG boxes in HMG1 and 2 for a variety of DNA targets, including four-way junctions, but not distorted DNA minicircles, to which the proteins bind with very high affinity. HMG1 and 2 appear to play important architectural roles in the assembly of nucleoprotein complexes in a variety of biological processes, for example V(D)J recombination, the initiation of transcription, and DNA repair.
TL;DR: A review of the literature of universal bases (to 2000) details the analogues investigated, and their uses and limitations are discussed.
Abstract: A universal base analogue forms ‘base pairs’ with each of the natural DNA/RNA bases with little discrimination between them A number of such analogues have been prepared and their applications as biochemical tools investigated Most of these analogues are non-hydrogen bonding, hydrophobic, aromatic ‘bases’ which stabilise duplex DNA by stacking interactions This review of the literature of universal bases (to 2000) details the analogues investigated, and their uses and limitations are discussed
TL;DR: A novel sequence-selective pyrrolobenzodiazepine (PBD) dimer 5 (SJG-136) has been developed that comprises two C2-exo-methylene-substituted DC-81 (3) subunits tethered through their C8 positions via an inert propanedioxy linker that is a highly efficient minor groove interstrand DNA cross-linking agent that is 440-fold more potent than melphalan.
Abstract: A novel sequence-selective pyrrolobenzodiazepine (PBD) dimer 5 (SJG-136) has been developed that comprises two C2-exo-methylene-substituted DC-81 (3) subunits tethered through their C8 positions via an inert propanedioxy linker. This symmetric molecule is a highly efficient minor groove interstrand DNA cross-linking agent (XL50 = 0.045 muM) that is 440-fold more potent than melphalan, Thermal denaturation studies show that, after 18 h incubation with calf thymus DNA at a 5:1 DNA/ligand ratio, it increases the T-m value by 33.6 degreesC, the highest value so far recorded in this assay. The analogous dimer 4 (DSB-120) that lacks substitution/ unsaturation at the C2 position elevates melting by only 15.1 degreesC under the same conditions, illustrating the effect of introducing C2-exo-unsaturation which serves to flatten the C-rings and achieve a superior isohelical fit within the DNA minor groove. This behavior is supported by molecular modeling studies which indicate that (i) the PBD units are covalently bonded to guanines on opposite strands to form a cross-link, (ii) 5 has a greater binding energy compared to 4, and (iii) 4 and 5 have equivalent binding sites that span six base pairs. Dimer 5 is significantly more cytotoxic than 4 in a number of human ovarian cancer cell lines (e.g., IC50 values of 0.0225 nM vs 7.2 nM, respectively, in A2780 cells). Furthermore, it retains full potency in the cisplatin-resistant cell line A2780cisR (0.024 nM), whereas 4 loses activity (0.21 muM) with a resistance factor of 29.2, This may be due to a lower level of inactivation of 5 by intracellular thiol-containing molecules. A dilactam analogue (21) of 5 that lacks the electrophilic N10-C11/N10'-C11' imine moieties has also been synthesized and evaluated. Although unable to interact covalently with DNA, 21 still stabilizes the helix (DeltaT(m) = 0.78 degreesC) and has significant cytotoxicity in some cell lines (i.e., IC50 = 0.57 muM in CH1 cells), presumably exerting its effect through noncovalent interaction with DNA.
TL;DR: The chloroplast chromosome of spinach is a double-stranded circular DNA molecule of 150 725 nucleotide pairs and a comparison of this chromosome with those of the three other autotrophic dicotyledons for which complete DNA sequences of plastid chromosomes are available confirms a conserved overall structure.
Abstract: The chloroplast chromosome of spinach (Spinacia oleracea) is a double-stranded circular DNA molecule of 150 725 nucleotide pairs. A comparison of this chromosome with those of the three other autotrophic dicotyledons for which complete DNA sequences of plastid chromosomes are available confirms a conserved overall structure. Three classes of open reading frames were distinguished: (1) genes of known function which include 108 unique loci, (2) three hypothetical chloroplast reading frames (ycfs) that are highly conserved interspecifically, and (3) species-specific or rapidly diverging 'open reading frames'. A detailed transcript study of one of the latter (ycf15) shows that these loci may be transcribed, but do not constitute protein-coding genes.
TL;DR: Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation.
TL;DR: The 2.0 Å crystal structure of the N6-adenine DNA methyltransferase M•TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base.
Abstract: The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.
TL;DR: This work has solved the crystal structure of a duplex containing the metallo-base pair and provides a structural explanation for the B- to Z-DNA transition in this duplex.
Abstract: Stable and selective DNA base pairing by metal coordination was recently demonstrated with nucleotides containing complementary pyridine-2,6-dicarboxylate (Dipic) and pyridine (Py) bases (Meggers, E.; Holland, P. L.; Tolman; W. B.; Romesberg, F. E.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 10714−10715). To understand the structural consequences of introducing this novel base pair into DNA we have solved the crystal structure of a duplex containing the metallo-base pair. The structure shows that the bases pair as designed, but in a Z-DNA conformation. The structure also provides a structural explanation for the B- to Z-DNA transition in this duplex. Further solution studies demonstrate that the metallo-base pair is compatible with Z- or B-DNA conformations, depending on the duplex sequence.