Showing papers on "Base pair published in 2006"

Structural basis for double-stranded RNA processing by Dicer.

Abstract: The specialized ribonuclease Dicer initiates RNA interference by cleaving double-stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.

MercuryII-mediated formation of thymine-HgII-thymine base pairs in DNA duplexes

Abstract: The very specific binding of the HgII ion unexpectedly and significantly stabilizes naturally occurring thymine−thymine base mispairing in DNA duplexes. Following this finding, we prepared DNA dupl...

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Abstract: Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A*T- and G*C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A*T pairing is always destabilizing and G*C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.

Analysis of one million base pairs of Neanderthal DNA

Abstract: Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. We have identified a 38,000-year-old Neanderthal fossil that is exceptionally free of contamination from modern human DNA. Direct high-throughput sequencing of a DNA extract from this fossil has thus far yielded over one million base pairs of hominoid nuclear DNA sequences. Comparison with the human and chimpanzee genomes reveals that modern human and Neanderthal DNA sequences diverged on average about 500,000 years ago. Existing technology and fossil resources are now sufficient to initiate a Neanderthal genome-sequencing effort.

Direct force measurements on DNA in a solid-state nanopore

Abstract: Among the variety of roles for nanopores in biology, an important one is enabling polymer transport, for example in gene transfer between bacteria1 and transport of RNA through the nuclear membrane2. Recently, this has inspired the use of protein3,4,5 and solid-state6,7,8,9,10 nanopores as single-molecule sensors for the detection and structural analysis of DNA and RNA by voltage-driven translocation. The magnitude of the force involved is of fundamental importance in understanding and exploiting this translocation mechanism, yet so far it has remained unknown. Here, we demonstrate the first measurements of the force on a single DNA molecule in a solid-state nanopore by combining optical tweezers11 with ionic-current detection. The opposing force exerted by the optical tweezers can be used to slow down and even arrest the translocation of the DNA molecules. We obtain a value of 0.24±0.02 pN mV−1 for the force on a single DNA molecule, independent of salt concentration from 0.02 to 1 M KCl. This force corresponds to an effective charge of 0.50±0.05 electrons per base pair equivalent to a 75% reduction of the bare DNA charge.

RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

Abstract: Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 +/- 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 +/- 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.

Genome-wide prediction of G4 DNA as regulatory motifs: Role in Escherichia coli global regulation

Abstract: The role of nonlinear DNA in replication, recombination, and transcription has become evident in recent years. Although several studies have predicted and characterized regulatory elements at the sequence level, very few have investigated DNA structure as regulatory motifs. Here, using G-quadruplex or G4 DNA motifs as a model, we have researched the role of DNA structure in transcription on a genome-wide scale. Analyses of >61,000 open reading frames (ORFs) across 18 prokaryotes show enrichment of G4 motifs in regulatory regions and indicate its predominance within promoters of genes pertaining to transcription, secondary metabolite biosynthesis, and signal transduction. Based on this, we predict that G4 DNA may present regulatory signals. This is supported by conserved G4 motifs in promoters of orthologous genes across phylogenetically distant organisms. We hypothesized a regulatory role of G4 DNA during supercoiling stress, when duplex destabilization may result in G4 formation. This is in line with our observations from target site analysis for 55 DNA-binding proteins in Escherichia coli, which reveals significant (P 1000 genes in the early growth phase and are believed to be induced by supercoiled DNA. We also predict G4 motif-induced supercoiling sensitivity for >30 operons in E. coli, and our findings implicate G4 DNA in DNA-topology-mediated global gene regulation in E. coli.

Partition function and base pairing probabilities of RNA heterodimers.

Abstract: RNA has been recognized as a key player in cellular regulation in recent years. In many cases, non-coding RNAs exert their function by binding to other nucleic acids, as in the case of microRNAs and snoRNAs. The specificity of these interactions derives from the stability of inter-molecular base pairing. The accurate computational treatment of RNA-RNA binding therefore lies at the heart of target prediction algorithms. The standard dynamic programming algorithms for computing secondary structures of linear single-stranded RNA molecules are extended to the co-folding of two interacting RNAs. We present a program, RNAcofold, that computes the hybridization energy and base pairing pattern of a pair of interacting RNA molecules. In contrast to earlier approaches, complex internal structures in both RNAs are fully taken into account. RNAcofold supports the calculation of the minimum energy structure and of a complete set of suboptimal structures in an energy band above the ground state. Furthermore, it provides an extension of McCaskill's partition function algorithm to compute base pairing probabilities, realistic interaction energies, and equilibrium concentrations of duplex structures. RNAcofold is distributed as part of the Vienna RNA Package, http://www.tbi.univie.ac.at/RNA/ . Stephan H. Bernhart – berni@tbi.univie.ac.at

APOBEC3G DNA deaminase acts processively 3' --> 5' on single-stranded DNA.

Abstract: Akin to a 'Trojan horse,' APOBEC3G DNA deaminase is encapsulated by the HIV virion. APOBEC3G facilitates restriction of HIV-1 infection in T cells by deaminating cytosines in nascent minus-strand complementary DNA. Here, we investigate the biochemical basis for C --> U targeting. We observe that APOBEC3G binds randomly to single-stranded DNA, then jumps and slides processively to deaminate target motifs. When confronting partially double-stranded DNA, to which APOBEC3G cannot bind, sliding is lost but jumping is retained. APOBEC3G shows catalytic orientational specificity such that deamination occurs predominantly 3' --> 5' without requiring hydrolysis of a nucleotide cofactor. Our data suggest that the G --> A mutational gradient generated in viral genomic DNA in vivo could result from an intrinsic processive directional attack by APOBEC3G on single-stranded cDNA.

A nanoplasmonic molecular ruler for measuring nuclease activity and DNA footprinting.

Abstract: Interactions between nucleic acids and proteins are essential to genetic information processing. The detection of size changes in nucleic acids is the key to mapping such interactions, and usually requires substrates with fluorescent, electrochemical or radioactive labels1,2,3. Recently, methods have been developed to tether DNA to highly water-soluble Au nanoparticles4,5,6,7,8, and nanoparticle pairs linked by DNA have been used to measure nanoscale distances9. Here we demonstrate a molecular ruler in which double-stranded DNA is attached to a Au nanoparticle. The change in plasmon resonance wavelength of individual Au–DNA conjugates depends on the length of the DNA and can be measured with subnanometre axial resolution. An average wavelength shift of approximately 1.24 nm is observed per DNA base pair. This system allows for a label-free, quantitative, real-time measurement of nuclease activity and also serves as a new DNA footprinting platform, which can accurately detect and map the specific binding of a protein to DNA.

Retroviral DNA integration: reaction pathway and critical intermediates.

Abstract: The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.

Local RNA base pairing probabilities in large sequences

Abstract: Summary: The genome-wide search for non-coding RNAs requires efficient methods to compute and compare local secondary structures. Since the exact boundaries of such putative transcripts are typically unknown, arbitrary sequence windows have to be used in practice. Here we present a method for robustly computing the probabilities of local base pairs from long RNA sequences independent of the exact positions of the sequence window. Availability: The program RNAplfold is part of the Vienna RNA Package and can be downloaded from http://www.tbi.univie.ac.at/RNA/ Contact: ivo@tbi.univie.ac.at

Fluorescent DNA base replacements: Reporters and sensors for biological systems.

Abstract: We describe the design, synthesis, and properties of nucleoside monomers in which the DNA base is replaced by fluorescent hydrocarbons and heterocycles, and the assembly of these monomers into DNA-like molecules in which the all bases are fluorescent. As monomers, such molecules have useful applications as reporters in the DNA context. The use of fluorescent DNA bases, rather than more traditional fluorophores tethered to the DNA strand, gives a more predictable location and orientation, and yields a more direct response to changes that occur within the helix. In addition to uses as monomers, such compounds can be assembled into polychromophoric oligomers (“oligodeoxyfluorosides” or ODFs). ODFs are water soluble, discrete molecules and are easily arranged into specific sequences by use of a DNA synthesizer. They have displayed a number of properties not readily available in commercial fluorophores, including large Stokes shifts, tunable excitation and emission wavelengths, and sensing responses to physical changes or molecular species in solution. We describe an approach to assembling and screening large sets of oligofluorosides for rapid identification of molecules with desirable properties. Such compounds show promise for applications in biochemistry, biology, environmental and materials applications.

An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA

Abstract: Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions. In replication, this unnatural base pair exhibits high selectivity in combination with the usual triphosphates and modified triphosphates, gamma-amidotriphosphates, as substrates of 3' to 5' exonuclease-proficient DNA polymerases, allowing PCR amplification. In transcription, the unnatural base pair complementarity mediates the incorporation of these base substrates and their analogs, such as a biotinylated substrate, into RNA by T7 RNA polymerase (RNAP). With this system, functional components can be site-specifically incorporated into a large RNA molecule.

A protocol for TILLING and Ecotilling in plants and animals

Abstract: We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.

Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb).

Abstract: The formation of G-quadruplex and i-motif structures in the 5' end of the retinoblastoma (Rb) gene was examined using chemical modifications, circular dichroism (CD) and fluorescence spectroscopy. It was found that substitutions of 8-methylguanine at positions that show syn conformations in antiparallel G-quadruplexes stabilize the structure in the G-rich strand. The complementary C-rich 18mer forms an i-motif structure, as suggested by CD spectroscopy. Based on the C to T mutation experiments, C bases participated in the C-C+ base pair of the i-motif structure were determined. Experiments of 2-aminopurine (2-AP) substitution reveal that an increase of fluorescence in the G-quadruplex relative to duplex is attributed to unstacked 2-AP within the loop of G-quadruplex. The fluorescence experiments suggest that formation of the G-quadruplex and i-motif can compete with duplex formation. Furthermore, a polymerase arrest assay indicated that formation the G-quadruplex structure in the Rb gene acts as a barrier in DNA synthesis.

Structure of a DNA glycosylase searching for lesions.

Abstract: DNA glycosylases must interrogate millions of base pairs of undamaged DNA in order to locate and then excise one damaged nucleobase. The nature of this search process remains poorly understood. Here we report the use of disulfide cross-linking (DXL) technology to obtain structures of a bacterial DNA glycosylase, MutM, interrogating undamaged DNA. These structures, solved to 2.0 angstrom resolution, reveal the nature of the search process: The protein inserts a probe residue into the helical stack and severely buckles the target base pair, which remains intrahelical. MutM therefore actively interrogates the intact DNA helix while searching for damage.

Direct Reversal of DNA Alkylation Damage

Abstract: 1.1. Overview of Direct Repair of DNA Alkylation Damage Cellular DNA is constantly subjected to modifications by intracellular and extracellular chemicals, which can result in covalent changes. 1,2 Alkylating agents are one group of such chemicals that can lead to DNA damage.3 These agents are prevalent in the environment and are used as anticancer compounds in the clinical setting.4–10 Alkylating agents also exist endogenously inside cells; for instance, S-adenosylmethionine, a methyl donor for many cellular reactions, has been shown to produce methylation damage.11,12 The attack on DNA by these alkylating agents can lead to various types of lesions on the heterocyclic bases or backbone.3,13–15 Most of these resulting adducts are mutagenic or toxic, and cells have evolved various proteins to detect and repair them.9,16,17 Interestingly, many of these alkylation lesions are repaired through the direct removal of the adduct. Other than the photolyase that catalyzes direct reversal of the thymine dimer created by UV light,18,19 all known direct DNA repair proteins are engaged in alkylation DNA damage repair. These are the N-terminal domain of the Escherichia coli (E. coli) Ada protein, the O6-alkylguanine-DNA alkyltransferase family, and the AlkB family.9 1.1.1. Alkylation of DNA Alkylating reagents can be divided into SN1 and SN2 types based on the mechanism of the alkylation attack. The alkylation susceptibility of each site on the bases or backbone varies depending on the reagent used (Figure 1); the resulting lesions also have different mutagenic and cytotoxic effects. The N7-position of guanine is the most vulnerable site on DNA; unsurprisingly, it also serves as the best ligand on the DNA for metal ions such as platinum(II).20 Treating double-stranded DNA (dsDNA) with methylating agents such as methylmethane sulfonate (MMS, an SN2 type methylating agent) or N-methyl-N′-nitrosourea (MNU, an SN1 type methylating agent) typically results in 70–80% of the methylation occurring on the N7-position of guanine. Despite being the most abundant product of alkylation damage, N7-methylguanine is relatively innocuous and is removed mostly through spontaneous depurination.21 The resulting abasic site is toxic and repaired enzymatically.22 The N3-methyladenine is the second most abundant alkylation lesion formed in dsDNA. This lesion can block DNA replication and is removed by AlkA in E. coli and 3-methyladenine-DNA-glycosylases.23–25 Figure 1 Methylation patterns of the DNA bases and phosphate backbone with MMS and MNU. The blue arrows indicate methylation sites that are repaired by glycosylases; the red arrows are for sites repaired by O6-alkylguanine-DNA alkyltransferases; the purple arrows ... The SN1 type methylating agents such as MNU and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) are highly mutagenic because they attack the oxygen atoms on DNA bases to give a significant amount of O6-methylguanine (O6-meG) and a small amount of O4-methylthymine (Figure 1).13,14 O6-meG mispairs with thymine during DNA replication, which gives rise to a transition mutation of G:C to A:T.26–29 Thus, this lesion must be rapidly located and removed in order to maintain the integrity of the genome. The O6-alkylguanine-DNA alkyltransferase family of proteins performs this important task in almost all organisms.29–33 The SN2 type methylating agents such as MMS and methyl halides can react with single-stranded DNA (ssDNA) to generate large portions of N1-methyladenine and N3-methylcytosine (Figure 1).3,9,13,14 These two positions are protected by hydrogen bonding in dsDNA but are quite nucleophilic when exposed in ssDNA or replication forks. When these sites are exposed, they are vulnerable to nucleophilic attack; the pKa’s of these two nitrogen sites are 4.1 and 4.5 in ssDNA, which are higher than that of N7-guanine.34–38 The resulting lesions prevent formation of Watson–Crick base pairs which could be toxic for cells. The protein involved in the repair of these lesions has been revealed only very recently. A family of iron(II)-dependent dioxygenases was found to catalytically remove these alkylation lesions.9,39,40 The phosphodiester DNA backbone is also subject to alkylation damage. For instance, 17% of the total methylation occurs on the backbone to yield methylphosphotriesters when dsDNA is treated with MNU (Figure 1). The neutral phosphotriester can be hydrolyzed by water much faster than the diester, which leads to cleavage of the backbone. The Sp-methylphosphotriester is repaired by the N-terminal domain of the Ada protein (N-Ada) in E. coli.17,41 This repair serves mostly as a signaling pathway to induce expression of methylation resistance genes, as will be discussed below. The other diastereomer, Rp-methylphosphotriester, cannot be repaired by N-Ada. There is no homologue of N-Ada found in eukaryotes. It is unclear whether methylphosphotriester is repaired in eukaryotes.

Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern

Abstract: To support efforts to develop a ‘synthetic biology’ based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3(1 0 -b-D-2 0 -deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson–Crick complement, the purine analog 2-amino-8-(1 0 -b-D-2 0 -deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where ‘py’ indicates a pyrimidine analog and ‘pu’ indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

On the Mechanism of Gene Amplification Induced under Stress in Escherichia coli

Abstract: Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s) of amplification in this system has been obtained, and many models for mechanism(s) were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications), potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7–32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3′-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging-strand templates are involved. Finally, we propose a novel, long-distance template-switching model for the mechanism of adaptive amplification that suggests how stress induces the amplifications. We outline its possible applicability to amplification in humans and other organisms and circumstances.

Stepwise translocation of Dpo4 polymerase during error-free bypass of an oxoG lesion

Abstract: 7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG•C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain–DNA phosphate contacts translocate by one nucleotide step, while the thumb domain–DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain–phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.