Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution

Sequence saturation mutagenesis (SeSaM) is a conceptually novel and practically simple method that truly randomizes a target sequence at every single nucleotide position. A SeSaM experiment can be accomplished within 2–3 days and comprises four steps: generating a pool of DNA fragments with random length, ‘tailing’ the DNA fragments with universal base using terminal transferase at 3′-termini, elongating DNA fragments in a PCR to the full-length genes using a single-stranded template and replacing the universal bases by standard nucleotides. Random mutations are created at universal sites due to the promiscuous base-pairing property of universal bases. Using enhanced green fluorescence protein as the model system and deoxyinosine as the universal base, we proved by sequencing 100 genes the concept of the SeSaM method and achieved a random distribution of mutations with the mutational bias expected for deoxyinosine.     

Universal bases for hybridization, replication and chain termination

Several unnatural, predominantly hydrophobic nucleobases that pack efficiently in duplex DNA without hydrogen bonding functionalities are reported to circumvent the hydrogen bonding-based specificity, both during oligonucleotide hybridization and enzymatic DNA synthesis. The reported nucleoside analogs are efficient ‘universal bases’ for hybridization, template directed DNA synthesis and chain termination. Moreover, several of the universal bases function in their biological role, hybridization or replication, with an efficiency not significantly reduced relative to their natural counterparts.     

DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study

We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the –10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the –10 and –35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions –15 and –14. Promoter activity is greatest when the ‘non-template’ strand carries T and G at positions –15 and –14, respectively. Promoter activity can be further enhanced by a second T and G at positions –17 and –16, respectively, immediately upstream of the first ‘TG motif’. Our results show that the base sequence of the DNA segment upstream of the –10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of ‘TG motifs’ upstream of the promoters’ –10 elements. We conclude that correctly placed ‘TG motifs’ are found at over 20% of E.coli promoters.     

Duplex-Repair enables highly accurate sequencing,despite DNA damage

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via ‘End Repair/dA-Tailing,’ may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7–17% and 32–57% of interior ‘duplex base pairs’ from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles’ heel in sequencing and offers a solution to restore high accuracy.     

Formation of an intramolecular triple-stranded DNA structure monitored by fluorescence of 2-aminopurine or 6-methylisoxanthopterin

The parallel (recombination) ‘R-triplex’ can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the fluorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or 6MI. The 2AP*(T·A) and 6MI*(C·G) triplets were monitored by their fluorescence emission and the thermal denaturation curves were analyzed with a quasi-two-state model. The fluorescence of 2AP introduced into an oligonucleotide sequence unable to form a triplex served as a negative control. We observed a remarkable similarity between the thermodynamic parameters derived from melting of the secondary structures monitored through absorption of all bases at 260 nm or from fluorescence of the single base analog. The similarity suggests that fluorescence of the 2AP and 6MI base analogs may be used to monitor the structural disposition of the third strand. We consider the data in the light of alternative ‘branch migration’ and ‘strand exchange’ structures and discuss why these are less likely than the R-type triplex.     

Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide

Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson–Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases.     

Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level

To assess whether there are universal rules that govern amino acid–base recognition, we investigate hydrogen bonds, van der Waals contacts and water-mediated bonds in 129 protein–DNA complex structures. DNA–backbone interactions are the most numerous, providing stability rather than specificity. For base interactions, there are significant base–amino acid type correlations, which can be rationalised by considering the stereochemistry of protein side chains and the base edges exposed in the DNA structure. Nearly two-thirds of the direct read-out of DNA sequences involves complex networks of hydrogen bonds, which enhance specificity. Two-thirds of all protein–DNA interactions comprise van der Waals contacts, compared to about one-sixth each of hydrogen and water-mediated bonds. This highlights the central importance of these contacts for complex formation, which have previously been relegated to a secondary role. Although common, water-mediated bonds are usually non-specific, acting as space-fillers at the protein–DNA interface. In conclusion, the majority of amino acid–base interactions observed follow general principles that apply across all protein–DNA complexes, although there are individual exceptions. Therefore, we distinguish between interactions whose specificities are ‘universal’ and ‘context-dependent’. An interactive Web-based atlas of side chain–base contacts provides access to the collected data, including analyses and visualisation of the three-dimensional geometry of the interactions.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.     

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔH_ITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex.     

Tethering-facilitated DNA ‘opening’ and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.     

A common perceptual temporal limit of binding synchronous inputs across different sensory attributes and modalities

The human brain processes different aspects of the surrounding environment through multiple sensory modalities, and each modality can be subdivided into multiple attribute-specific channels. When the brain rebinds sensory content information (‘what’) across different channels, temporal coincidence (‘when’) along with spatial coincidence (‘where’) provides a critical clue. It however remains unknown whether neural mechanisms for binding synchronous attributes are specific to each attribute combination, or universal and central. In human psychophysical experiments, we examined how combinations of visual, auditory and tactile attributes affect the temporal frequency limit of synchrony-based binding. The results indicated that the upper limits of cross-attribute binding were lower than those of within-attribute binding, and surprisingly similar for any combination of visual, auditory and tactile attributes (2–3 Hz). They are unlikely to be the limits for judging synchrony, since the temporal limit of a cross-attribute synchrony judgement was higher and varied with the modality combination (4–9 Hz). These findings suggest that cross-attribute temporal binding is mediated by a slow central process that combines separately processed ‘what’ and ‘when’ properties of a single event. While the synchrony performance reflects temporal bottlenecks existing in ‘when’ processing, the binding performance reflects the central temporal limit of integrating ‘when’ and ‘what’ properties.     

Rational design of DNA sequences for nanotechnology, microarrays and molecular computers using Eulerian graphs

Nucleic acids are molecules of choice for both established and emerging nanoscale technologies. These technologies benefit from large functional densities of ‘DNA processing elements’ that can be readily manufactured. To achieve the desired functionality, polynucleotide sequences are currently designed by a process that involves tedious and laborious filtering of potential candidates against a series of requirements and parameters. Here, we present a complete novel methodology for the rapid rational design of large sets of DNA sequences. This method allows for the direct implementation of very complex and detailed requirements for the generated sequences, thus avoiding ‘brute force’ filtering. At the same time, these sequences have narrow distributions of melting temperatures. The molecular part of the design process can be done without computer assistance, using an efficient ‘human engineering’ approach by drawing a single blueprint graph that represents all generated sequences. Moreover, the method eliminates the necessity for extensive thermodynamic calculations. Melting temperature can be calculated only once (or not at all). In addition, the isostability of the sequences is independent of the selection of a particular set of thermodynamic parameters. Applications are presented for DNA sequence designs for microarrays, universal microarray zip sequences and electron transfer experiments.     

Incoming nucleotide binds to Klenow ternary complex leading to stable physical sequestration of preceding dNTP on DNA

Klenow–DNA complex is known to undergo a rate-limiting, protein conformational transition from an ‘open’ to ‘closed’ state, upon binding of the ‘correct’ dNTP at the active site. In the ‘closed’ state, Mg²⁺ mediates a rapid chemical step involving nucleophilic displacement of pyrophosphate by the 3′ hydroxyl of the primer terminus. The enzyme returns to the ‘open’ state upon the release of PPi and translocation permits the next round of reaction. To determine whether Klenow can translocate to the next site on the addition of the next dNTP, without the preceding chemical step, we studied the ternary complex (Klenow–DNA–dNTP) in the absence of Mg²⁺. While the ternary complex is proficient in chemical addition of dNTPs in Mg²⁺, as revealed by primer extensions, the same in Mg²⁺-deficient conditions lead to non-covalent (physical) sequestration of first two ‘correct’ dNTPs in the ternary complex. Moreover, the second dNTP traps the first one in the DNA-helix of the ternary complex. Such a dNTP–DNA complex is found to be stable even after the dissociation of Klenow. This reveals the novel state of the dNTP–DNA complex where the complementary base is stacked in a DNA-helix non-covalently, without the phosphodiester linkage. Further, shuttling of the DNA between the polymerase and the exonuclease site mediates the release of such a DNA complex. Interestingly, Klenow in such a Mg²⁺-deficient ternary complex exhibits a ‘closed’ conformation.     

Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions

The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the ‘between gene’ GC content heterogeneity, which is linked to ‘isochores’, is a principal factor associated with the bias in substitution patterns in human, ‘within gene’ heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed.     

Focus: Sex and Gender Health: Patient Preference for Physician Gender in the Emergency Department

Despite historical gender bias against female physicians, few studies have investigated patients’ physician gender preference in the emergency department (ED) setting. We sought to determine if there is an association between ED patient demographics and physician gender preference. We surveyed patients presenting to an ED to determine association between patient demographics and patient physician gender preference for five ED situations: 1) ‘routine’ visit, 2) emergency visit, 3) ‘sensitive’ medical visit, 4) minor surgical/‘procedural’ visit, and 5) ‘bad news’ delivery. A total of 200 ED patients were surveyed. The majority of ED patients reported no physician gender preference for ‘routine’ visits (89.5 percent), ‘emergent’ visits (89 percent), ‘sensitive’ medical visits (59 percent), ‘procedural’ visits (89 percent) or when receiving ‘bad news’ (82 percent). In the setting of ‘routine’ visits and ‘sensitive’ medical visits, there was a propensity for same-sex physician preference.     

Specificity of Mnt 'master residue' obtained from in vivo and in vitro selections

Mnt is a repressor from phage P22 that belongs to the ribbon–helix–helix family of DNA binding factors. Four amino acids from the N-terminus of the protein, Arg2, His6, Asn8 and Arg10, interact with the base pairs of the DNA to provide the sequence specificity. Raumann et al. (Nature Struct. Biol., 2, 1115–1122) identified position 6 as a ‘master residue’ that controls the specificity of the protein. Models for the interaction have residue 6 of Mnt interacting directly with position 5 of the operator. In vivo selections demonstrated that protein variants at residue 6 bound specifically to operator mutations at that position. Operators in which the wild-type G at position 5 was replaced by T specifically bound to several different protein variants, primarily hydrophobic residues. The obtained protein variants, plus some others, were used in in vitro selections to determine their preferred binding sites. The results showed that the residue at position 6 influenced the preference for binding site bases predominantly at position 5, but that the effects of altering it can extend over longer distances, consistent with its designation as a ‘master residue’. The similarities of binding sites for different residues do not correlate strongly with common measures of amino acid similarities.     

Communicating and Dealing with Uncertainty in General Practice: The Association with Neuroticism

Background

Diagnostic reasoning in primary care setting where presented problems and patients are mostly unselected appears as a complex process. The aim was to develop a questionnaire to describe how general practitioners (GPs) deal with uncertainty to gain more insight into the decisional process. The association of personality traits with medical decision making was investigated additionally.

Methods

Raw items were identified by literature research and focus group. Items were improved by interviewing ten GPs with thinking-aloud-method. A personal case vignette related to a complex and uncertainty situation was introduced. The final questionnaire was administered to 228 GPs in Germany. Factorial validity was calculated with explorative and confirmatory factor analysis. The results of the Communicating and Dealing with Uncertainty (CoDU) – questionnaire were compared with the scales of the ‘Physician Reaction to Uncertainty’ (PRU) questionnaire and with the personality traits which were determined with the Big Five Inventory (BFI-K).

Results

The items could be assigned to four scales with varying internal consistency, namely ‘communicating uncertainty’ (Cronbach alpha 0.79), ‘diagnostic action’ (0.60), ‘intuition’ (0.39) and ‘extended social anamnesis’ (0.69). Neuroticism was positively associated with all PRU scales ‘anxiety due to uncertainty’ (Pearson correlation 0.487), ‘concerns about bad outcomes’ (0.488), ‘reluctance to disclose uncertainty to patients’ (0.287), ‘reluctance to disclose mistakes to physicians’ (0.212) and negatively associated with the CoDU scale ‘communicating uncertainty’ (−0.242) (p<0.01 for all). ‘Extraversion’ (0.146; p<0.05), ‘agreeableness’ (0.145, p<0.05), ‘conscientiousness’ (0.168, p<0.05) and ‘openness to experience’ (0.186, p<0.01) were significantly positively associated with ‘communicating uncertainty’. ‘Extraversion’ (0.162), ‘consciousness’ (0.158) and ‘openness to experience’ (0.155) were associated with ‘extended social anamnesis’ (p<0.05).

Conclusion

The questionnaire allowed describing the diagnostic decision making process of general practitioners in complex situations. Personality traits are associated with diagnostic reasoning and communication with patients, which might be important for medical education and quality improvement purposes.     

Gamma Oscillations as a Neural Signature of Shifting Times in Narrative Language

Verbs and other temporal expressions allow speakers to specify the location of events in time, as well as to move back and forth in time, shifting in a narrative between past, present and future. The referential flexibility of temporal expressions is well understood in linguistics but its neurocognitive bases remain unknown. We aimed at obtaining a neural signature of shifting times in narrative language. We recorded and analyzed event-related brain potentials (ERPs) and oscillatory responses to the adverb ‘now’ and to the second main verb in Punctual (‘An hour ago the boy stole a candy and now he peeled the fruit’) and Iterative (‘The entire afternoon the boy stole candy and now he peeled the fruit’) contexts. ‘An hour ago’ introduces a time frame that lies entirely in the past, ‘now’ shifts the narrative to the present, and ‘peeled’ shifts it back to the past. These two referential shifts in Punctual contexts are expected to leave very similar traces on neural responses. In contrast, ‘The entire afternoon’ specifies a time frame that may encompass past, present and future, such that both ‘now’ and ‘peeled’ are consistent with it. Here, no time shift is required. We found no difference in ERPs between Punctual and Iterative contexts either at ‘now’ or at the second verb. However, reference shifts modulated oscillatory signals. ‘Now’ and the second verb in Punctual contexts resulted in similar responses: an increase in gamma power with a left-anterior distribution. Gamma bursts were absent in Iterative contexts. We propose that gamma oscillations here reflect the binding of temporal variables to the values allowed by constraints introduced by temporal expressions in discourse.     

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational ‘hot-spots’ or ‘cold-spots’. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.