The theoretical limits of DNA sequence discrimination by linked polyamides

Linked polyamides bind in the minor groove of double-stranded DNA in a partially sequence-specific manner. This report analyzes the theoretical limits of DNA sequence discrimination by linked polyamides composed of two to four different types of heterocyclic rings, determining (i) the optimal choice of base-binding specificity for each ring and (ii) the optimal design for a polyamide composed of these rings to target a given DNA sequence and designed to maximize the fraction of the total polyamide binding to the specified target sequence relative to all other sequences. The results show that, fortuitously, polyamides composed of pyrrole, a naturally occurring G-excluding element, and imidazole, a rationally designed G-favoring element, have features similar to the theoretical optimum design for polyamides composed of two different rings. The results also show that, in polyamides composed of two or three types of heterocyclic rings, choosing a nonspecific “placeholder” ring, which binds equally strongly to each of the four bases, along with one or two base-specific rings will often enhance sequence specificity over a polyamide composed entirely of base-specific rings.     

Application of a time-dependent coalescence process for inferring the history of population size changes from DNA sequence data

Distribution of pairwise differences of nucleotides from data on a sample of DNA sequences from a given segment of the genome has been used in the past to draw inferences about the past history of population size changes. However, all earlier methods assume a given model of population size changes (such as sudden expansion), parameters of which (e.g., time and amplitude of expansion) are fitted to the observed distributions of nucleotide differences among pairwise comparisons of all DNA sequences in the sample. Our theory indicates that for any time-dependent population size, N(τ) (in which time τ is counted backward from present), a time-dependent coalescence process yields the distribution, p(τ), of the time of coalescence between two DNA sequences randomly drawn from the population. Prediction of p(τ) and N(τ) requires the use of a reverse Laplace transform known to be unstable. Nevertheless, simulated data obtained from three models of monotone population change (stepwise, exponential, and logistic) indicate that the pattern of a past population size change leaves its signature on the pattern of DNA polymorphism. Application of the theory to the published mtDNA sequences indicates that the current mtDNA sequence variation is not inconsistent with a logistic growth of the human population.     

Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.     

Relative binding affinities of monovalent cations for double-stranded DNA

The competition between sodium and various other monovalent cations that bind to helical DNA in aqueous solution has been studied by ²³Na NMR. Variations in the sodium linewidth with the concentration of the other ion have been analyzed with an equation that describes the competitive binding in terms of two parameters: r, the total extent of counterion binding, and D, a measure of the binding affinity of a cation relative to sodium. The concentration dependence of these parameters was found to be minimal. In the absence of a competing cation the constancy of r has been demonstrated over a range of DNA phosphate concentrations (0.0025-0.015 M) and NaCl concentrations (0.003-1.3 M). For the cations investigated the range in D values is small (0.5-0.9), and the relative binding affinities follow the order: NH₄⁺ > Cs⁺ > K⁺ > Li⁺ > Na⁺.     

Right-handed double-helix ultrashort DNA yields chiral nematic phases with both right- and left-handed director twist

Concentrated solutions of duplex-forming DNA oligomers organize into various mesophases among which is the nematic (N^∗), which exhibits a macroscopic chiral helical precession of molecular orientation because of the chirality of the DNA molecule. Using a quantitative analysis of the transmission spectra in polarized optical microscopy, we have determined the handedness and pitch of this chiral nematic helix for a large number of sequences ranging from 8 to 20 bases. The B-DNA molecule exhibits a right-handed molecular double-helix structure that, for long molecules, always yields N^∗ phases with left-handed pitch in the μm range. We report here that ultrashort oligomeric duplexes show an extremely diverse behavior, with both left- and right-handed N^∗ helices and pitches ranging from macroscopic down to 0.3 μm. The behavior depends on the length and the sequence of the oligomers, and on the nature of the end-to-end interactions between helices. In particular, the N^∗ handedness strongly correlates with the oligomer length and concentration. Right-handed phases are found only for oligomers shorter than 14 base pairs, and for the sequences having the transition to the N^∗ phase at concentration larger than 620 mg/mL. Our findings indicate that in short DNA, the intermolecular double-helical interactions switch the preferred liquid crystal handedness when the columns of stacked duplexes are forced at high concentrations to separations comparable to the DNA double-helix pitch, a regime still to be theoretically described.     

Cyclic polyamides for recognition in the minor groove of DNA.

Small molecules that specifically bind with high affinity to any designated DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine. Simple rules have been developed to rationally alter the sequence specificity of minor groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Crescent-shaped polyamides bind as antiparallel dimers with each polyamide making specific contacts with each strand on the floor of the minor groove. Cyclic polyamides have now been synthesized that bind designated DNA sequences at subnanomolar concentrations.     

Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K_d < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have ∼3–8-fold higher affinity and ∼2–4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.     

Induction of microsatellite instability by oxidative DNA damage

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 10⁶ wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N′-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.     

The catalytic domain of λ site-specific recombinase

The Escherichia coli phage λ integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that λ Int consists of three functional and structural domains: residues 1–64 specify recognition of “arm-type” DNA sequences distant from the region of strand exchange; residues 65–169 contribute to specific recognition of “core-type” sequences at the sites of strand exchange and possibly to protein–protein interactions; and residues 170–356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.     

Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol⁻¹, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol⁻¹ and T_m raised by 5.6°C is of potential interest for trials in vivo.     

Specificity in cholesterol regulation of gene expression by coevolution of sterol regulatory DNA element and its binding protein

When demand for cholesterol rises in mammalian cells, the sterol regulatory element (SRE) binding proteins (SREBPs) are released from their membrane anchor through proteolysis. Then, the N-terminal region enters the nucleus and activates genes of cholesterol uptake and biosynthesis. Basic helix–loop–helix (bHLH) proteins such as SREBPs bind to a palindromic DNA sequence called the E-box (5′-CANNTG-3′). However, SREBPs are special because they also bind direct repeat elements called SREs. Importantly, sterol regulation of all promoters studied thus far is mediated by SREBP binding only to SREs. To study the reason for this we converted the direct repeat SRE from the sterol-regulated low-density lipoprotein receptor promoter into an E-box. In this report we show that SREBPs are still able to bind and activate this promoter however, sterol regulation is lost. The results are consistent with the mutant promoter being a target for promiscuous activation by constitutively expressed E-box binding bHLH proteins that are not regulated by cholesterol. Kim and coworkers [Kim, J. B., Spotts, G. D., Halvorsen, Y.-D., Shih, H.-M., Ellenberger, T., Towle, H. C. & Spiegelman, B. M. (1995) Mol. Cell. Biol. 15, 2582–2588] demonstrated that the dual DNA binding specificity of SREBPs is caused by a specific tyrosine in the conserved basic region of the DNA binding domain that corresponds to an arginine in all other bHLH proteins that recognize only E-boxes. Taken together the data suggest an evolutionary mechanism where a DNA binding protein along with its recognition site have coevolved to ensure maximal specificity and sensitivity in a crucial nutritional regulatory response.     

The domain organization of NaeI endonuclease: Separation of binding and catalysis

NaeI is a remarkable type II restriction endonuclease. It must bind two recognition sequences to cleave DNA, forms a covalent protein–DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity. The latter activities apparently derive from reactivation of a cryptic DNA ligase active site. Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa. The domains were purified by cloning. The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI. Analytical ultracentrifugation showed this domain to be a monomer in solution. The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association. DNA greatly inhibited proteolysis of the linker region. The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI.     

Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2^82–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer.     

How clonal are Neisseria species? The epidemic clonality model revisited

The three species Neisseria meningitidis, Neisseria gonorrheae, and Neisseria lactamica are often regarded as highly recombining bacteria. N. meningitidis has been considered a paradigmatic case of the “semiclonal model” or of “epidemic clonality,” demonstrating occasional bouts of clonal propagation in an otherwise recombining species. In this model, occasional clonality generates linkage disequilibrium in the short term. In the long run, however, the effects of clonality are countered by recombination. We show that many data are at odds with this proposal and that N. meningitidis fits the criteria that we have proposed for predominant clonal evolution (PCE). We point out that (i) the proposed way to distinguish epidemic clonality from PCE may be faulty and (ii) the evidence of deep phylogenies by microarrays and whole-genome sequencing is at odds with the predictions of the semiclonal model. Last, we revisit the species status of N. meningitidis, N. gonorrheae, and N. lactamica in the light of the PCE model.     

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

Bacterial DNA replication requires DnaA, an AAA+ ATPase that initiates replication at a specific chromosome region, oriC, and is regulated by species-specific regulators that directly bind DnaA. HobA is a DnaA binding protein, recently identified as an essential regulator of DNA replication in Helicobacter pylori. We report the crystal structure of HobA in complex with domains I and II of DnaA (DnaA^I–II) from H. pylori, the first structure of DnaA bound to one of its regulators. Biochemical characterization of the complex formed shows that a tetramer of HobA binds four DnaA^I–II molecules, and that DnaA^I–II is unable to oligomerize by itself. Mutagenesis and protein–protein interaction studies demonstrate that some of the residues located at the HobA-DnaA^I–II interface in the structure are necessary for complex formation. Introduction of selected mutations into H. pylori shows that the disruption of the interaction between HobA and DnaA is lethal for the bacteria. Remarkably, the DnaA binding site of HobA is conserved in DiaA from Escherichia coli, suggesting that the structure of the HobA/DnaA complex represents a model for DnaA regulation in other Gram-negative bacteria. Our data, together with those from other studies, indicate that HobA could play a crucial scaffolding role during the initiation of replication in H. pylori by organizing the first step of DnaA oligomerization and attachment to oriC.