Mixed DNA Sequences Research Articles

Single GC base pair recognition by a heterocyclic diamidine: structures, affinities, and dynamics.

The recognition of specific genomic arrangements by rationally designed small molecules is fundamental for the expansion of targeted gene expression. Here, we report the first X-ray crystal structures that demonstrate single G (guanine) recognition by a highly selective diamidine (DB2447) in a mixed DNA sequence. The study presents detailed structural information on the mechanism of single G recognition by D2447 and its various interactions in the DNA minor groove. Molecular dynamics and binding studies were used to evaluate the details of our reported structures. The study provides structural insight and resources necessary for understanding single G selection in genomic sequences.

X-ray Structure Characterization of the Selective Recognition of AT Base Pair Sequences.

The rational design of small molecules that target specific DNA sequences is a promising strategy to modulate gene expression. This report focuses on a diamidinobenzimidazole compound, whose selective binding to the minor groove of AT DNA sequences holds broad significance in the molecular recognition of AT-rich human promoter sequences. The objective of this study is to provide a more detailed and systematized understanding, at an atomic level, of the molecular recognition mechanism of different AT-specific sequences by a rationally designed minor groove binder. The specialized method of X-ray crystallography was utilized to investigate how the sequence-dependent recognition properties in general, A-tract, and alternating AT sequences affect the binding of diamidinobenzimidazole in the DNA minor groove. While general and A-tract AT sequences give a narrower minor groove, the alternating AT sequences intrinsically have a wider minor groove which typically constricts upon binding. A strong and direct hydrogen bond between the N-H of the benzimidazole and an H-bond acceptor atom in the minor groove is essential for DNA recognition in all sequences described. In addition, the diamidine compound specifically utilizes an interfacial water molecule for its DNA binding. DNA complexes of AATT and AAAAAA recognition sites show that the diamidine compound can bind in two possible orientations with a preference for water-assisted hydrogen bonding at either cationic end. The complex structures of AAATTT, ATAT, ATATAT, and AAAA are bound in a singular orientation. Analysis of the helical parameters shows a minor groove expansion of about 1 Å across all the nonalternating DNA complexes. The results from this systematic approach will convey a greater understanding of the specific recognition of a diverse array of AT-rich sequences by small molecules and more insight into the design of small molecules with enhanced specificity to AT and mixed DNA sequences.

Systematic synthetic and biophysical development of mixed sequence DNA binding agents.

It is now well established that, although only about 5% of the human genome codes for protein, most of the DNA has some function, such as synthesis of specific, functional RNAs and/or control of gene expression. These functional sequences open immense possibilities in both biotechnology and therapeutics for the use of cell-permeable, small molecules that can bind mixed-base pair sequences of DNA for regulation of genomic functions. Unfortunately very few types of modules have been designed to recognize mixed DNA sequences and for progress in targeting specific genes, it is essential to have additional classes of compounds. Compounds that can be rationally designed from established modules and which can bind strongly to mixed base pair DNA sequences are especially attractive. Based on extensive experience in design of minor-groove agents for AT recognition, a small library of compounds with two AT specific binding modules, connected through linkers which can recognize the G·C base pairs, were prepared. The compound-DNA interactions were evaluated with a powerful array of biophysical methods and the results show that some pyridyl-linked compounds bind with the target sequence with sub-nanomolar KD, with very slow dissociation kinetics and 200 times selectivity over the related sequence without a G·C base pair. Interestingly, a set of compounds with AT module connected by different linkers shows cooperative dimer recognition of related sequences. This type of design approach can be expanded to additional modules for recognition of a wide variety of sequences.

Emergence of Life from Trapped Nucleotides? Non-Equilibrium ­Behavior of Oligonucleotides in Thermal Gradients

How life emerged is one of the major questions that remains to be answered. Apart from being of interest for completeness of biology, it is also a very interesting study case from the vantage point of physics. Living organisms are inherently non-equilibrium systems. Since non-equilibrium thermodynamics is still a developing field, the emergence of life is a highly interesting study case. Here we present the progress we have made during the last few years, employing experimental biophysics to capture the mechanisms that could eventually lead to the emergence of life. We show how a simple non-equilibrium system, a thermal gradient, gives rise to a range of relevant phenomena, in particular, polymerization, elongation, and replication of DNA molecules as well as demixing of mixed DNA sequences into sequence-pure hydrogels. 1 Introduction 2 Thermophoresis, the Biased Movement of Molecules in Thermal Gradients 3 The Dynamical Behavior of Accumulated Molecules 4 Overcoming Spiegelman’s Monster 5 Sequence Purification and Oscillations by a Gel-Phase Transition in Thermal Gradients 6 Loose Ends 7 Discussion and Conclusion

Legal, Technical, and Interpretational Considerations in the Forensic Analysis of Viruses

The forensic evaluation of viruses presents new challenges to the forensic science community. Although many criminal cases have been adjudicated involving the deliberate transmission of viruses, especially HIV, this review provides a general approach to viral forensics, especially in light of significant biodefense challenges. Newly emerging techniques of nucleic acid sequencing are discussed in a forensic context. Human mitochondrial DNA analysis, wherein mixed profiles are routinely assessed in a forensic context, provides the groundwork for an interpretational approach to the issue of mixed DNA sequences. The importance of phylogenetic classification is discussed as both providing an integrated graphical depiction of the structure of viral nucleic acid variation as well as offering a tool that can be used to assess the relatedness of complex populations of nucleic acids.

Blobology: exploring raw genome data for contaminants, symbionts and parasites using taxon-annotated GC-coverage plots

Generating the raw data for a de novo genome assembly project for a target eukaryotic species is relatively easy. This democratization of access to large-scale data has allowed many research teams to plan to assemble the genomes of non-model organisms. These new genome targets are very different from the traditional, inbred, laboratory-reared model organisms. They are often small, and cannot be isolated free of their environment – whether ingested food, the surrounding host organism of parasites, or commensal and symbiotic organisms attached to or within the individuals sampled. Preparation of pure DNA originating from a single species can be technically impossible, but assembly of mixed-organism DNA can be difficult, as most genome assemblers perform poorly when faced with multiple genomes in different stoichiometries. This class of problem is common in metagenomic datasets that deliberately try to capture all the genomes present in an environment, but replicon assembly is not often the goal of such programs. Here we present an approach to extracting, from mixed DNA sequence data, subsets that correspond to single species’ genomes and thus improving genome assembly. We use both numerical (proportion of GC bases and read coverage) and biological (best-matching sequence in annotated databases) indicators to aid partitioning of draft assembly contigs, and the reads that contribute to those contigs, into distinct bins that can then be subjected to rigorous, optimized assembly, through the use of taxon-annotated GC-coverage plots (TAGC plots). We also present Blobsplorer, a tool that aids exploration and selection of subsets from TAGC-annotated data. Partitioning the data in this way can rescue poorly assembled genomes, and reveal unexpected symbionts and commensals in eukaryotic genome projects. The TAGC plot pipeline script is available from https://github.com/blaxterlab/blobology, and the Blobsplorer tool from https://github.com/mojones/Blobsplorer.

Dependence of DNA–Protein Cross-Linking via Guanine Oxidation upon Local DNA Sequence As Studied by Restriction Endonuclease Inhibition

Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.

Detection of T-T Mismatches Using Mass Spectrometry: Specific Interactions of Hg(II) with Oligonucleotides Rich in Thymine (T)

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed in a detailed study of the interactions of mercury dications with selected oligodeoxynucleotides rich and poor in thymine (T): d(5'-TT-3'), d(5'-TTT-3'), d(5'-TTTT-3'), d(5'-GG-3'), d(5'-CC-3'), d(5'-AA-3'), d(5'-GCTTGC-3'), d(5'-GTGCTC-3'), d(5'-GCATGC-3'), and d(5'-GCGCGC-3'). Specific interactions are observed for Hg(2+) with pure and mixed thymine sequences in which simultaneous bonding between two thymine units is indicated, and this is consistent with a model proposed in the literature in which Hg(2+) covalently coordinates to two thymines by replacing two N3 imino protons of the bases. The ESI-MS/MS measurements, combined with data on the thermal stability of mixed sequence hexadeoxynucleotides, indicate that mercury prefers thymines over the other binding sites in oligonucleotides both in solution and in the gas phase. These results point toward the effective use of Hg(2+) in the fast detection of mismatch base pairs incorporated in oligonucleotide duplexes and longer mixed DNA sequences using ESI-MS/MS.

Ability of Polymerase η and T7 DNA Polymerase to Bypass Bulge Structures

DNA misalignment occurs in homopolymer tracts during replication and can lead to frameshift mutations. Polymerase (pol) recognition of primer-templates containing bulge structures and the transmission of a bulge through a polymerase binding site or replication complex are important components of frameshift mutagenesis. In this report, we describe the interaction of the catalytic core of pol eta with primer-templates containing bulge structures by single round primer extension. We found that pol eta could stabilize a frayed primer terminus, which enhances its ability to extend primer-templates containing bulges. Based on methylphosphonate-DNA mapping, pol eta interacts with the single strand template but not appreciably with the template strand of the DNA stem greater than two nucleotides from the primer terminus. These latter characteristics, combined with the ability to stabilize a frayed primer terminus, may explain why primer-templates containing template bulges are extended so efficiently by pol eta. Although pol eta could accommodate large bulges and continue synthesis without obstruction, bulge structures in the template, but not in the primer, caused termination of the T7 DNA replication complex. Terminations occurred when the template bulge neared the helix-loop-helix domain of the polymerase thumb. Terminations were not observed, however, when bulge structures approached the site of interaction of the DNA with the extended thumb and thioredoxin. At low temperature, however, terminations did occur at this site.

Effect of DNA sequence and nucleotide cofactors on hRad51 binding to ssDNA: Role of hRad52 in recruitment

hRad51 binding to ssDNA is significantly lowered in the presence of a nucleotide cofactor ATP/ADP/ATPγS. In these conditions, presence of trace amounts of hRad52 protein restores hRad51 binding to DNA. In the absence of any nucleotide cofactor where intrinsic binding of hRad51 to ssDNA is higher, hRad52 brings about no improved binding. hRad51 binding to ssDNA is strongly influenced by the DNA sequence. The protein binding to repeat sequences is poor compared to that of mixed DNA sequence. Interestingly, presence of hRad52 restores the ability of hRad51 binding to such DNA targets as well. Moreover, all the cooperative effects of hRad52 on hRad51 binding are highly specific to the latter’s binding to ssDNA and not to dsDNA. These results help us to model important mechanistic steps of hRad51 presynapsis on ssDNA templates.

The effect of varied ion distributions on long-range DNA charge transport.

Long-range oxidative damage to DNA was utilized as a probe to delineate the effects of different ion distributions on DNA charge transport. DNA assemblies were constructed, containing a tethered rhodium intercalating photooxidant, spatially separated from two 5'-GG-3' sites of oxidative damage, with either an A6-tract or a mixed DNA sequence intervening between the guanine doublets; the extent of charge transport was assessed through measurements of the ratio of yields of damage at the guanine doublet distal versus that proximal to the metal binding site. The distal/proximal damage ratios were compared after photooxidation of otherwise identical Rh-tethered assemblies, except for 32P-labeling either at the 5'- or 3'-end; this labeling difference corresponds, in the absence of charge neutralization by condensed counterions, to a shift in negative charge from one end of the duplex to the other. Both with assemblies containing the mixed sequence and the A6-tract, we observed that moving the negative charges to the proximal end of the duplex significantly decreased hole transport to the distal end. We propose that these results reflect variations in the thermodynamic potential at the proximal and distal guanine sites because of the change in charges at the termini of the oligomer. High values for the internal dielectric constant of the stacked base pairs are suggested by these data. Hence, the longitudinal polarizability of DNA may be important to consider in mechanisms for long-range DNA charge transport.

Specific molecular recognition of mixed nucleic acid sequences: an aromatic dication that binds in the DNA minor groove as a dimer.

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

On the recombinational origin of protein-sequence-subunit structure.

Since 1929 the concept that proteins are built from subunits of certain standard size (Svedberg 1929) has been revisited several times, each time with a new demonstration that, indeed, there are certain preferred protein sizes. According to recent estimates the overrepresented sizes are close to multiples of 125 amino acid (aa) residues for eukaryotes and 150 residues for prokaryotes. To explain these preferences, a hypothesis is suggested, and quantitatively developed, on the recombinational nature of this regularity. The protein-coding sequences are assumed to evolve at some early stage via recombinational events--insertions of DNA circles of a certain optimal size. The contour lengths of the protein-coding DNA circles had to be simultaneously divisible by three and, to minimize torsional constraint, by the DNA helical repeat. With these two conditions satisfied, the calculated contour lengths of the DNA circles, 250-500 base pairs (bp), turn out to correspond well to known optimal DNA circularization sizes and to the predicted range of the protein sequence subunit sizes: 80-170 aa residues, which covers experimentally observed values. The subunit size is found to be strongly influenced by the helical repeat of DNA. The sizes 125 and 150 aa are derived when the corresponding helical repeats of DNA are set within fractions of promilles from the 10.54 bp/turn value. This fits to the experimentally estimated mean for natural mixed DNA sequences, 10.53-10.57 bp/turn.(ABSTRACT TRUNCATED AT 250 WORDS)