Local DNA Structure Research Articles

DNA conformation in nanochannels: Monte Carlo simulation studies using a primitive DNA model

We have performed canonical ensemble Monte Carlo simulations of a primitive DNA model to study the conformation of 2.56 ~ 21.8 μm long DNA molecules confined in nanochannels at various ionic concentrations with the comparison of our previous experimental findings. In the model, the DNA molecule is represented as a chain of charged hard spheres connected by fixed bond length and the nanochannels as planar hard walls. System potentials consist of explicit electrostatic potential along with short-ranged hard-sphere and angle potentials. Our primitive model system provides valuable insight into the DNA conformation, which cannot be easily obtained from experiments or theories. First, the visualization and statistical analysis of DNA molecules in various channel dimensions and ionic strengths verified the formation of locally coiled structures such as backfolding or hairpin and their significance even in highly stretched states. Although the folding events mostly occur within the region of ~0.5 μm from both chain ends, significant portion of the events still take place in the middle region. Second, our study also showed that two controlling factors such as channel dimension and ionic strength widely used in stretching DNA molecules have different influence on the local DNA structure. Ionic strength changes local correlation between neighboring monomers by controlling the strength of electrostatic interaction (and thus the persistence length of DNA), which leads to more coiled local conformation. On the other hand, channel dimension controls the overall stretch by applying the geometric constraint to the non-local DNA conformation instead of directly affecting local correlation. Third, the molecular weight dependence of DNA stretch was observed especially in low stretch regime, which is mainly due to the fact that low stretch modes observed in short DNA molecules are not readily accessible to much longer DNA molecules, resulting in the increase in the stretch of longer DNA molecules.

Colloidal Structure and Stability of DNA/Polycations Polyplexes Investigated by Small Angle Scattering

Polyplexes of short DNA-fragments (300 b.p., 100 nm) with tailor-made amine-based polycations of different architectures (linear and hyperbranched) were investigated in buffer solution as a function of the mixing ratio with DNA. The resulting dispersed polyplexes were characterized using small-angle neutron and X-ray scattering (SANS, SAXS) as well as cryo-TEM with respect to their mesoscopic structure and their colloidal stability. The linear polyimines form rather compact structures that have a high tendency for precipitation. In contrast, the hyperbranched polycation with enzymatic-labile pentaethylenehexamine arms (PEHA) yields polyplexes colloidally stable for months. Here the polycation coating of DNA results in a homogeneous dispersion based on a fractal network with low structural organization at low polycation amount. With increasing polycation, bundles of tens of aligned DNA rods appear that are interconnected in a fractal network with a typical correlation distance on the order of 100 nm, the average length of the DNA used. With higher organization comes a decrease in stability. The 3D network built by these beams can still exhibit some stability as long as the material concentration is large enough, but the structure collapses upon dilution. SAXS shows that the complexation does not affect the local DNA structure. Interestingly, the structural findings on the DNA polyplexes apparently correlate with the transfection efficiency of corresponding siRNA complexes. In general, these finding not only show systematic trends for the colloid stability, but may allow for rational approaches to design effective transfection carriers.

Base-Flipping Mechanism in Postmismatch Recognition by MutS

DNA mismatch recognition and repair is vital for preserving the fidelity of the genome. Conserved across prokaryotes and eukaryotes, MutS is the primary protein that is responsible for recognizing a variety of DNA mismatches. From molecular dynamics simulations of the Escherichia coli MutS-DNA complex, we describe significant conformational dynamics in the DNA surrounding a G·T mismatch that involves weakening of the basepair hydrogen bonding in the basepair adjacent to the mismatch and, in one simulation, complete base opening via the major groove. The energetics of base flipping was further examined with Hamiltonian replica exchange free energy calculations revealing a stable flipped-out state with an initial barrier of ∼2 kcal/mol. Furthermore, we observe changes in the local DNA structure as well as in the MutS structure that appear to be correlated with base flipping. Our results suggest a role of base flipping as part of the repair initiation mechanism most likely leading to sliding-clamp formation.

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA+) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA+ domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA+ domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.

High nuclease activity of a copper(II)-bipyridyl complex containing cytosine pendants

A new copper(II) complex bearing 2,2′-bipyridine (bpy) derivatives with cytosine nucleobases [Cu(L)Cl2] · 2H2O has been synthesized and characterized, where L = 5,5′-di[(4-amino-2-oxo-1(2H)-pyrimidinyl)methyl]-2,2′-bipyridine. Circular dichroism spectra reveal interactions between ligand and pBR322 DNA and showed that the local DNA structure was perturbed by the ligand. Cleavage of pBR322 DNA by the title complex was carried out in 20 mmol L−1 HEPES (pH 7.5) at 37°C. The calculated pseudo-Michaelis–Menten kinetic parameter (k cat) of the complex was 8.80 h−1. The cleavage efficiency was 17-fold higher than that of its simple analog [Cu(bpy)Cl2] (k cat = 0.50 h−1). Hydrolytic mechanisms involved in DNA cleavage were explored using radical scavengers and T4 ligase. Competitive experiments with special binding agents showed that the complex could preferentially bind to the minor groove of double-stranded DNA, suggesting specific DNA binding characteristics. High DNA cleavage reactivity and selectivity of the complex could be attributed to the synergic effects of the metal center and the cytosine pendants.

An effective approach to artificial nucleases using copper(ii) complexes bearing nucleobases

Novel copper(ii) complexes bearing 2,2'-bipyridine (bpy) derivatives with adenine, thymine and uracil nucleobases [Cu(L(1))Cl(2)].2H(2)O (1), [Cu(L(2))Cl(2)] (2) and [Cu(L(3))Cl(2)].H(2)O (3) (L(1) = 5,5'-Di[N9-adenylmethyl]-2,2'-bipyridine, L(2) = 5,5'-Di[N1-thyminylmethyl]-2,2'-bipyridine and L(3) = 5,5'-Di[N1-uracilmethyl]-2,2'-bipyridine) were synthesized and characterized. Structure simulation was performed for these complexes. Circular dichroism (CD) spectra revealed the interactions between these ligands and pBR322 DNA and showed that the local DNA structure was perturbed by these ligands. Cleavage of pBR322 DNA by these complexes was carried out in 20 mM HEPES (pH 7.5) at 37 degrees C. The calculated pseudo-Michaelis-Menten kinetic parameters (k(cat)) were 14.7 +/- 0.6 and 40.4 +/- 1.3 h(-1) for and . The cleavage efficiency of was 80-fold higher than that of its simple analogue [Cu(bpy)Cl(2)] (k(cat) = 0.50 h(-1)) and very close to the catalytic rate constant of natural EcoRI endonuclease (k(cat) = 43.2 h(-1)) at similar conditions. Thus, complex might be one of the most effective artificial nucleases that could catalyze double-stranded DNA hydrolytic cleavage so far. Hydrolytic mechanisms involved in DNA cleavage were explored using radical scavengers and T4 ligase. Competitive experiments with special binding agents showed that complexes could preferentially bind to the minor groove of double-stranded DNA, suggesting specific DNA binding characteristics. Molecular docking calculations also indicated that complexes could bind to the minor groove of targeted DNA much more strongly than their simple analogues and preferentially bind at the AT region of the dodecamer. Such high DNA cleavage ability and selectivity of these copper(ii) complexes could be attributed to the synergic effects of the metal center and the pendant nucleobases.

Horizontally acquired homologues of the nucleoid‐associated protein H‐NS: implications for gene regulation

H-NS is one of the most intensively studied members of the family of bacterial nucleoid-associated proteins. It is a DNA-binding protein with a preference for A+T-rich DNA sequences, and it represses the transcription of hundreds of genes in Gram-negative bacteria, including pathogens. In most cases where the issue has been investigated, the repressive activity of H-NS is opposed by the intervention of an antagonistically acting DNA-binding protein, a remodelling of local DNA structure, or a combination of these two. H-NS activity can also be modulated by protein-protein interaction with members of the Hha/YdgT protein family, molecules that share partial amino acid sequence similarity to the oligomerization domain of H-NS. Of particular interest is the ability of H-NS to interact with the full-length paralogue StpA or full-length orthologues that have been acquired by horizontal DNA transfer. In this issue of Molecular Microbiology, Müller et al. describe the H-NS orthologue Hfp and present evidence that in bacteria that acquire Hfp the range of activities of H-NS is modified with important implications for the physiology of the bacterium.

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

alpha-Accessory factor (AAF) stimulates the activity of DNA polymerase-alpha.primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44.AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-alpha.primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-(3)H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-alpha.primase template affinity and stimulate both DNA primase and polymerase-alpha activities in vitro.

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

The H-NS nucleoid-associated DNA-binding protein is an important global repressor of transcription in Gram-negative bacteria. Recently, H-NS has been implicated in the process of xenogeneic silencing, where it represses the transcription of foreign genes acquired by horizontal transfer. This raises interesting questions about the integration of the horizontally acquired genes into the existing gene regulatory networks of the microbe. In particular, how do bacteria derepress silenced genes in order to benefit from their expression without compromising competitive fitness through doing so inappropriately? This article reviews current knowledge about the derepression of genes that are transcriptionally silenced by H-NS. It describes a variety of anti-silencing mechanisms involving (i) protein-independent processes that operate at the level of local DNA structure, (ii) DNA-binding proteins such as Ler, LeuO, RovA, SlyA, VirB, and proteins related to AraC, and (iii) modulatory mechanisms in which H-NS forms heteromeric protein-protein complexes with full-length or partial paralogues such as StpA, Sfh, Hha, YdgT, YmoA or H-NST. The picture that emerges is one of apparently ad hoc solutions to the problem of H-NS-mediated silencing, suggesting that microbes are capable of evolving anti-silencing methods based on the redeployment of existing regulatory proteins rather than employing dedicated, bespoke antagonists. There is also evidence that in a number of cases more sophisticated regulatory processes have been superimposed on these rather simple anti-silencing mechanisms, broadening the range of environmental signals to which H-NS-repressed genes respond.

Elongating RNA Polymerase II Is Disassembled through Specific Degradation of Its Largest but Not Other Subunits in Response to DNA Damage in Vivo

Although previous biochemical studies have demonstrated global degradation of the largest subunit, Rpb1p, of RNA polymerase II in response to DNA damage, it is still not clear whether the initiating or elongating form of Rpb1p is targeted for degradation in vivo. Further, whether other components of RNA polymerase II are degraded in response to DNA damage remains unknown. Here, we show that the Rpb1p subunit of the elongating, but not initiating, form of RNA polymerase II is degraded at the active genes in response to 4-nitroquinoline-1-oxide-induced DNA damage in Saccharomyces cerevisiae. However, other subunits of RNA polymerase II are not degraded in response to DNA damage. Further, we show that Rpb1p is essential for RNA polymerase II assembly at the active gene, and thus, the degradation of Rpb1p following DNA damage disassembles elongating RNA polymerase II. Taken together, our data demonstrate that Rpb1p but not other subunits of elongating RNA polymerase II is specifically degraded in response to DNA damage, and such a degradation of Rpb1p is critical for the disassembly of elongating RNA polymerase II at the DNA lesion in vivo.

Intrastrand Annealing Leads to the Formation of a Large DNA Palindrome and Determines the Boundaries of Genomic Amplification in Human Cancer

Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double-strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and nonrandomly distributed in the genomes of cancer cells and facilitate a further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known how a DSB is resolved to form a large DNA palindrome and whether any local DNA structure determines the location of large DNA palindromes. We show here that intrastrand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determine the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and nonamplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome.

Identification of insertion hot spots for non-LTR retrotransposons: computational and biochemical application to Entamoeba histolytica

The genome of the human pathogen Entamoeba histolytica contains non-long terminal repeat (LTR) retrotransposons, the EhLINEs and EhSINEs, which lack targeted insertion. We investigated the importance of local DNA structure, and sequence preference of the element-encoded endonuclease (EN) in selecting target sites for retrotransposon insertion. Pre-insertion loci were tested computationally to detect unique features based on DNA structure, thermodynamic considerations and protein interaction measures. Target sites could readily be distinguished from other genomic sites based on these criteria. The contribution of the EhLINE1-encoded EN in target site selection was investigated biochemically. The sequence-specificity of the EN was tested in vitro with a variety of mutated substrates. It was possible to assign a consensus sequence, 5′-GCATT-3′, which was efficiently nicked between A-T and T-T. The upstream G residue enhanced EN activity, possibly serving to limit retrotransposition in the A+T-rich E.histolytica genome. Mutated substrates with poor EN activity showed structural differences compared with normal substrates. Analysis of retrotransposon insertion sites from a variety of organisms showed that, in general, regions of favorable DNA structure were recognized for retrotransposition. A combination of favorable DNA structure and preferred EN nicking sequence in the vicinity of this structure may determine the genomic hotspots for retrotransposition.

Structural and dynamic basis of a supercoiling-responsive DNA element

In both eukaryotes and prokaryotes, negative supercoiling of chromosomal DNA acts locally to regulate a variety of cellular processes, such as transcription, replication, recombination and response to environmental stresses. While studying the interaction between the Hin recombinase and mutated versions of its cognate DNA-binding site, we identified a mutated DNA site that binds Hin only when the DNA is supercoiled. To understand the mechanism of this supercoiling-responsive DNA site, we used NMR spectroscopy and fluorescence resonance energy transfer to determine the solution structures and dynamics of three related DNA oligonucleotides. The supercoiling-responsive DNA site formed a partially unwound and stretched helix and showed significant flexibility and base pair opening kinetics. The single CAG/CTG triplet contained in this DNA sequence displayed the same characteristics as do multiple CAG/CTG repeats, which are associated with several hereditary neuromuscular diseases. It is known that short DNA sequence motifs that have either very high or low bending flexibility occur preferentially at supercoiling-sensitive bacterial and eukaryotic promoters. From our results and these previous data, we propose a model in which supercoiling utilizes the intrinsic flexibility of a short DNA site to switch the local DNA structure from an inefficient conformation for protein binding to an efficient one, or vice versa.

Structure of DNA Complexes with Nonhistone Chromosomal Protein HMGB1 in the Presence of Manganese Ions

The complexes of DNA–HMGB1 protein–manganese ions have been studied using the circular dichroism (CD) technique. It was shown that the interactions of both the protein and metal ions with DNA in this three-component system differ from those in two-component complexes. The manganese ions did not affect the CD spectrum of the free HMGB1 protein. However, Mn2+ ions induced considerable changes in the CD spectrum of free DNA in the spectral range of 260–290 nm. The presence of Mn2+ ions prevented the formation of the ordered supramolecular structures typical of HMGB1–DNA complexes. The interaction of manganese ions with DNA had a marked influence on the local DNA structure, changing the properties of protein-binding sites and resulting in a marked decrease in cooperativity of HMGB1–DNA binding. Such changes in the mode of protein–DNA interactions occurred at concentrations as small as 0.01 mM Mn2+. Moreover, the changes in local DNA structure induced by the manganese ions promoted the appearance of new HMGB1 binding sites in the DNA double helix. At the same time, interactions with the HMGB1 protein induced alterations in the structure of the DNA double helix, which increased with an increase in the protein-to-DNA ratio. These alterations made the DNA–protein complex especially sensitive to manganese ions. Under these conditions the Mn2+ ions strongly affected the DNA structure, which was reflected in abrupt changes in the CD spectra of DNA in the complex in the range of 260–290 nm. Thus, structural changes in the DNA double helix in three-component DNA–HMGB1–Mn2+ complexes result from the combined and interdependent interactions of DNA with Mn2+ ions and HMGB1 molecules.

Evidence for glycosidic bond rotation in a nucleobase peroxyl radical and its effect on tandem lesion formation.

Nucleobase peroxyl radicals are the major reactive intermediates formed in DNA when the biopolymer is exposed to gamma-radiolysis under aerobic conditions. The major reaction pathways for the peroxyl radical (1) derived from 5,6-dihydro-2'-deoxyuridin-6-yl involve pi-bond addition to or hydrogen atom abstraction from the adjacent nucleotides to produce tandem lesions. The ability to independently generate 1 at a defined site in DNA enabled us to probe its reactivity by varying the local DNA structure. The effect of DNA structure variation reveals that 1 reacts from its syn- and anti-conformations in competition with trapping by thiol. These experiments also reveal that tandem lesions will be produced as a mixture of diastereomers, which could impact their biological effects.

Molecular dynamics simulation study of oriented polyamine- and Na-DNA: sequence specific interactions and effects on DNA structure.

Molecular dynamics (MD) computer simulations have been carried out on four systems that correspond to an infinite array of parallel ordered B-DNA, mimicking the state in oriented DNA fibers and also being relevant for crystals of B-DNA oligonucleotides. The systems were all comprised of a periodical hexagonal cell with three identical DNA decamers, 15 water molecules per nucleotide, and counterions balancing the DNA charges. The sequence of the double helical DNA decamer was d(5'-ATGCAGTCAG)xd(5'-TGACTGCATC). The counterions were the two natural polyamines spermidine(3+) (Spd(3+)) and putrescine(2+) (Put(2+)), the synthetic polyamine diaminopropane(2+) (DAP(2+)), and the simple monovalent cation Na(+). This work compares the specific structures of the polyamine- and Na-DNA systems and how they are affected by counterion interactions. It also describes sequence-specific hydration and interaction of the cations with DNA. The local DNA structure is dependent on the nature of the counterion. Even the very similar polyamines, Put(2+) and DAP(2+), show clear differences in binding to DNA and in effect on hydration and local structure. Generally, the polyamines disorder the hydration of the DNA around their binding sites whereas Na(+) being bound to DNA attracts and organizes water in its vicinity. Cation binding at the selected sites in the minor and in the major groove is compared for the different polyamines and Na(+). We conclude that the synthetic polyamine (DAP(2+)) binds specifically to several structural and sequence-specific motifs on B-DNA, unlike the natural polyamines, Spd(3+) and Put(2+). This specificity of DAP(2+) compared to the more dynamic behavior of Spd(3+) and Put(2+) may explain why the latter polyamines are naturally occurring in cells.

Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli

Microarray analysis shows that transcription of 306 E. Coli genes is affected by changes in the level of chromosome supercoiling, suggesting that supercoiling transmits regulatory signals from the environment to many cellular pathways.

Cleavage of fragments containing DNA mismatches by enzymic and chemical probes.

We prepared synthetic 50-mer DNA duplexes, each containing four mismatched base-pairs in similar positions. We examined their cleavage by DNases I and II, micrococcal nuclease (MNase), methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] and hydroxyl radicals. We find that single mismatches only produce subtle changes in the DNase I-cleavage pattern, the most common of which is attenuated cleavage at locations 2-3 bases on the 3'-side of the mismatch. Subtle changes are also observed in most of the DNase II-cleavage patterns, although GT and GG inhibit the cleavage over longer regions and generate patterns that resemble footprints. MNase cleaves the heteroduplexes at the mismatches themselves (except for CC), and in some cases cleaves CpG and CpC steps. None of the mismatches causes any change in the cleavage patterns produced by hydroxyl radicals or MPE-Fe(II). We also examined the cleavage patterns of fragments containing tandem GA mismatches in the sequences RGAY/RGAY and YGAR/YGAR (R, purine; Y, pyrimidine). RGAY causes only subtle changes in the cleavage patterns, which are similar to those seen with single mismatches, except that there are no changes in MNase cleavage. However, YGAR inhibits DNases I and II cleavage over 4-6 bases, and attenuates MPE-Fe(II) and hydroxyl radical cleavage at 2 bases. These changes suggest that this mismatch has a more pronounced effect on the local DNA structure. These changes are discussed in terms of the structural and dynamic effects of each mismatch.

Global and Local DNA Structure and Dynamics. Single molecule studies with AFM

Global and Local DNA Structure and Dynamics. Single molecule studies with AFM

Alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) recognition of DNA: an NMR spectroscopic study.

We have used NMR and CD spectroscopy to study and characterise two alpha-L-LNA:DNA duplexes, a nonamer that incorporates three alpha-L-LNA nucleotides and a decamer that incorporates four alpha-L-LNA nucleotides, in which alpha-L-LNA is alpha-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson-Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H-1H NOE measurements. In general, the deoxyriboses in both of the alpha-L-LNA:DNA duplexes adopt S-type (B-type structure) sugar puckers, that is the inclusion of the modified alpha-L-LNA nucleotides does not perturb the local native B-like double-stranded DNA (dsDNA) structure. The CD spectra of the duplexes confirm these findings, as these display B-type characteristic features that allow us to characterise the overall duplex type as B-like. The 1H-1H NOE distances which were determined for the decamer duplex were employed in a simulated annealing protocol to generate a model structure for this duplex, thus allowing a more detailed inspection of the impact of the alpha-L-ribo-configured nucleotides. In this structure, it is evident that the malleable DNA backbone rearranges in the vicinity of the modified nucleotides in order to accommodate them and present their nucleobases in a geometry suitable for Watson-Crick base pairing.