Energy mapping of the genetic code and genomic domains: implications for code evolution and molecular Darwinism.

When the iconic DNA genetic code is expressed in terms of energy differentials, one observes that information embedded in chemical sequences, including some biological outcomes, correlate with distinctive free energy profiles. Specifically, we find correlations between codon usage and codon free energy, suggestive of a thermodynamic selection for codon usage. We also find correlations between what are considered ancient amino acids and high codon free energy values. Such correlations may be reflective of the sequence-based genetic code fundamentally mapping as an energy code. In such a perspective, one can envision the genetic code as composed of interlocking thermodynamic cycles that allow codons to 'evolve' from each other through a series of sequential transitions and transversions, which are influenced by an energy landscape modulated by both thermodynamic and kinetic factors. As such, early evolution of the genetic code may have been driven, in part, by differential energetics, as opposed exclusively by the functionality of any gene product. In such a scenario, evolutionary pressures can, in part, derive from the optimization of biophysical properties (e.g. relative stabilities and relative rates), in addition to the classic perspective of being driven by a phenotypical adaptive advantage (natural selection). Such differential energy mapping of the genetic code, as well as larger genomic domains, may reflect an energetically resolved and evolved genomic landscape, consistent with a type of differential, energy-driven 'molecular Darwinism'. It should not be surprising that evolution of the code was influenced by differential energetics, as thermodynamics is the most general and universal branch of science that operates over all time and length scales.

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion.

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype-phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction-dependent destabilization of the underlying double helix, and a self-structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self-structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size-dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes.

Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states.

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as "rollamers". Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain ("rollamerization"). We demonstrate that such "rollameric" migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor "rollamers" with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic "universal hinge" relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.

Energy crosstalk between DNA lesions: implications for allosteric coupling of DNA repair and triplet repeat expansion pathways.

Energy coupling between distal DNA domains may have profound regulatory consequences for biological processes, allowing for allosteric control of nucleic acid function. Repair of oxidative lesions at or near triplet repeat domains can enhance DNA expansion events that result in debilitating disease states. We report here position, distance, and lesion-dependent energy crosstalk between pairs of lesions in a triplet repeat bulge loop and an adjacent duplex domain. We discuss the implications of such coupled communication between lesions in distal loop and duplex domains for lesion repair and DNA expansion associated with diseases.

Energetic coupling between clustered lesions modulated by intervening triplet repeat bulge loops: allosteric implications for DNA repair and triplet repeat expansion.

Clusters of closely spaced oxidative DNA lesions present challenges to the cellular repair machinery. When located in opposing strands, base excision repair (BER) of such lesions can lead to double strand DNA breaks (DSB). Activation of BER and DSB repair pathways has been implicated in inducing enhanced expansion of triplet repeat sequences. We show here that energy coupling between distal lesions (8oxodG and/or abasic sites) in opposing DNA strands can be modulated by a triplet repeat bulge loop located between the lesion sites. We find this modulation to be dependent on the identity of the lesions (8oxodG vs. abasic site) and the positions of the lesions (upstream vs. downstream) relative to the intervening bulge loop domain. We discuss how such bulge loop-mediated lesion crosstalk might influence repair processes, while favoring DNA expansion, the genotype of triplet repeat diseases.

DNA repair and DNA triplet repeat expansion: the impact of abasic lesions on triplet repeat DNA energetics.

Enhanced levels of DNA triplet expansion are observed when base excision repair (BER) of oxidative DNA base damage (e.g., 8-oxo-dG) occurs at or near CAG repeat sequences. This observation suggests an interplay between processing mechanisms required for DNA repair and expansion pathways that yield genotypes associated with many neurological/developmental disorders. It has been proposed that DNA expansion involves the transient formation within the triplet repeat domains of non-native slipped DNA structures that are incorrectly processed by the BER machinery of repair during DNA synthesis. We show here that replacement within a triplet repeat bulge loop domain of a guanosine residue by an abasic site, the universal BER intermediate, increases the population of slipped/looped DNA structures relative to the corresponding lesion-free construct. Such abasic lesion-induced energetic enhancement of slipped/looped structures provides a linkage between BER and DNA expansion. We discuss how the BER machinery of repair may be influenced by abasic-induced energetic alterations in the properties of regions proximal to and/or within triplet repeat domains, thereby potentially modulating levels of DNA expansion.

DNA energy landscapes via calorimetric detection of microstate ensembles of metastable macrostates and triplet repeat diseases.

Biopolymers exhibit rough energy landscapes, thereby allowing biological processes to access a broad range of kinetic and thermodynamic states. In contrast to proteins, the energy landscapes of nucleic acids have been the subject of relatively few experimental investigations. In this study, we use calorimetric and spectroscopic observables to detect, resolve, and selectively enrich energetically discrete ensembles of microstates within metastable DNA structures. Our results are consistent with metastable, "native" DNA states being composed of an ensemble of discrete and kinetically stable microstates of differential stabilities, rather than exclusively being a single, discrete thermodynamic species. This conceptual construct is important for understanding the linkage between biopolymer conformational/configurational space and biological function, such as in protein folding, allosteric control of enzyme activity, RNA and DNA folding and function, DNA structure and biological regulation, etc. For the specific DNA sequences and structures studied here, the demonstration of discrete, kinetically stable microstates potentially has biological consequences for understanding the development and onset of DNA expansion and triplet repeat diseases.

DNA metastability and biological regulation: conformational dynamics of metastable omega-DNA bulge loops.

Dynamic interchange between DNA conformations, including metastable states, can be of importance to biological function. In this study, we use a combination of spectroscopic and calorimetric techniques to detect and characterize kinetically trapped, metastable states in strand exchange and strand displacement reactions for bulge loop DNA conformations, here referred to as Omega-DNAs. We show that such metastable, Omega-DNA bulge loop states can stably coexist below 50 degrees C, while rearranging irreversibly at elevated temperatures to thermodynamically more stable states. Such dynamic interchange between metastable and globally stable DNA conformational states can be of importance in biological regulatory mechanisms.

The energetics of i-DNA tetraplex structures formed intermolecularly by d(TC5) and intramolecularly by d[(C5T3)3C5].

Cytosine-rich DNA at low pH adopts an antiparallel tetraplex structure via the intercalation of two partially protonated, parallel stranded duplexes. This intriguing structural motif has been named i-DNA. We have used a combination of spectroscopic and calorimetric techniques to characterize the properties of an intermolecular i-DNA formed by d(TC(5)) and an intramolecular i-DNA formed by d[(C(5)T(3))(3)C(5)]. Our measurements reveal that both i-DNA complexes are enthalpically stabilized by 6.5-7.0 kcal/mol(base) and entropically destabilized by 20 cal/mol(base)/K. These values are about 50% larger than the corresponding enthalpy and entropy values per base for Watson and Crick duplexes and for Hoogsteen triplexes, while being similar to per base enthalpy and entropy values reported for G-quadruplexes. Our data also reveal a positive heat capacity change between 20 and 30 cal/mol(base)/K, values similar to that reported for polymeric Watson & Crick DNA duplexes. Solution-dependent studies reveal the overall thermal and thermodynamic stability of i-DNA complexes to be dictated by an interplay between pH and ionic strength. Based on the thermodynamic data measured, we discuss the feasibility of i-DNA formation in the context of conventional DNA sequences, while commenting on potential roles for this structural motif in biological regulatory mechanisms.

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism.

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

Length-dependent energetics of (CTG)n and (CAG)n trinucleotide repeats.

Trinucleotide repeats are involved in a number of debilitating diseases such as myotonic dystrophy. Twelve to seventy-five base-long (CTG)n oligodeoxynucleotides were analysed using a combination of biophysical [UV-absorbance, circular dichroism and differential scanning calorimetry (DSC)] and biochemical methods (non-denaturing gel electrophoresis and enzymatic footprinting). All oligomers formed stable intramolecular structures under near physiological conditions with a melting temperature that was only weakly dependent on oligomer length. Thermodynamic analysis of the denaturation process by UV-melting and calorimetric experiments revealed an unprecedented length-dependent discrepancy between the enthalpy values deduced from model-dependent (UV-melting) and model-independent (calorimetry) experiments. Evidence for non-zero molar heat capacity changes was also derived from the analysis of the Arrhenius plots and DSC profiles. Such behaviour is analysed in the framework of an intramolecular 'branched-hairpin' model, in which long CTG oligomers do not fold into a simple long hairpin-stem intramolecular structure, but allow the formation of several independent folding units of unequal stability. We demonstrate that, for sequences ranging from 12 to 25 CTG repeats, an intramolecular structure with two loops is formed which we will call 'bis-hairpin'. Similar results were also found for CAG oligomers, suggesting that this observation may be extended to various trinucleotide repeats-containing sequences.

Intercalation into the DNA double helix and in vivo biological activity of water-soluble planar [Pt(diimine)(N,N-dihydroxyethyl-N'-benzoylthioureato)]+Cl- complexes: a study of their thermal stability, their CD spectra and their gel mobility.

The interaction of newly synthesised water-soluble planar complexes of general structure [Pt(diimine)(N,N-dihydroxyethyl-N'-benzoylthioureato)]+Cl- with DNA was investigated by means of DNA melting studies, CD spectroscopy, and DNA gel mobility studies. Addition of stoichometric amounts of [Pt(diimine)H2L-S,O]Cl complexes to polynucleotides caused a significant increase in the melting temperature of poly(dA-dT) and calf-thymus DNA, respectively, indicating that these complexes interacted with DNA and stabilised the double helical structure. The CD spectra confirmed the relatively strong binding of three related Pt(II) complexes ([Pt(2,2'-bipyridine)H2L-S,O]Cl, [Pt(4,4'-dimethyl-2,2'-bipyridine)H2L-S,O]Cl, and [Pt(1,10-phenanthroline)H2L-S,O]Cl), to DNA. Comparison with the published CD spectra of ethidium bromide/DNA complex suggests a similar intercalation mode of binding. cis-[(4,4'-di-tert-butyl-2,2'-bipyridyl)N,N-di(2-hydroxyethyl)-N'-benzoylthioureatoplatinum(II)] chloride, with its very bulky tert-butyl groups, did not intercalate into the polynucleotide double helix. In DNA mobility studies in the presence of the four [Pt(diimine)H2L-S,O]Cl complexes, only [Pt(2,2'-bipyridine)H2L-S,O]Cl affected the DNA mobility to any detectable extent. Finally, in vivo studies on the biological activity of the complexes, using an Escherichia coli DNA excision repair deficient uvrA mutant strain, indicated that only the [Pt(2,2'-bipyridine)H2L-S,O]Cl complex showed significant cellular toxicity and that this was, in part, linked to DNA damage.

Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases.

The effects of various protoporphyrinogen oxidase (PPOX) mutations responsible for variegate porphyria (VP), the roles of the arginine-59 residue and the glycines in the conserved flavin binding site, in catalysis and/or cofactor binding, were examined. Wild-type recombinant human PPOX and a selection of mutants were generated, expressed, purified and partially characterised. All mutants had reduced PPOX activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind flavin. The positive charge at arginine-59 appears to be directly involved in catalysis and not in flavin-cofactor binding alone. The K(m)s for the arginine-59 mutants suggested a substrate-binding problem. T(1/2) indicated that arginine-59 is required for the integrity of the active site. The dominant alpha-helical content was decreased in the mutants. The degree of alpha-helix did not correlate linearly with T(1/2) nor T(m) values, supporting the suggestion that arginine-59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of glycine in codon 14 was less disruptive than substitutions in codons 9 and 11. Ultraviolet melting curves generally showed a two-state transition suggesting formation of a multi-domain structure. All mutants studied were more resistant to thermal denaturation compared to wild type, except for R168C.