Base Pair Opening Research Articles

The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme–substrate complex is not the bottleneck controlling the catalytic activity of AAG.

The thermodynamics and kinetics of a nucleotide base pair.

The thermodynamic and kinetic parameters of an RNA base pair were obtained through a long-time molecular dynamics simulation of the opening-closing switch process of the base pair near its melting temperature. The thermodynamic parameters were in good agreement with the nearest-neighbor model. The opening rates showed strong temperature dependence, however, the closing rates showed only weak temperature dependence. The transition path time was weakly temperature dependent and was insensitive to the energy barrier. The diffusion constant exhibited super-Arrhenius behavior. The free energy barrier of breaking a single base stack results from the enthalpy increase, ΔH, caused by the disruption of hydrogen bonding and base-stacking interactions. The free energy barrier of base pair closing comes from the unfavorable entropy loss, ΔS, caused by the restriction of torsional angles. These results suggest that a one-dimensional free energy surface is sufficient to accurately describe the dynamics of base pair opening and closing, and the dynamics are Brownian.

The Effect of Local Melting of DNA on DNA Loop Formation

Statistical mechanics of double-stranded DNA (dsDNA) is well described by the wormlike chain model (WLC) which assumes a harmonic bending potential. Such smooth bending potential may no longer be valid for large bending angles to form small loops (< 100 bp). Instead, DNA may rely on rare structural transitions such as local melting (opening) of base pairs to lower the energetic cost. In theory, open base pairs called bubbles can increase the looping probability of short DNA molecules by a few orders of magnitude, but a robust experimental validation of this theoretical prediction is lacking. Here, we investigated the correlation between local melting probability and looping dynamics of dsDNA using single-molecule fluorescence resonance energy transfer (FRET). We designed two groups of short DNA molecules with low and high melting probabilities around their center and measured their looping and unlooping rates in equilibrium. Our data allow rigorous tests of meltable wormlike chain (MWLC) models at short length scales for setting the lower limit for the free energy of bubble formation.

Pulling short DNA molecules having defects on different locations.

We present a study on the role of defects on the stability of short DNA molecules. We consider short DNA molecules (16 base pairs) and investigate the thermal as well as mechanical denaturation of these molecules in the presence of defects that occur anywhere in the molecule. For the investigation, we consider four different kinds of chains. Not only are the ratios of AT to GC different in these molecules but also the distributions of AT and GC along the molecule are different. With suitable modifications in the statistical model to show the defect in a pair, we investigate the denaturation of short DNA molecules in thermal as well as constant force ensembles. In the force ensemble, we pulled the DNA molecule from each end (keeping other end free) and observed some interesting features of opening of the molecule in the presence of defects in the molecule. We calculate the probability of opening of the DNA molecule in the constant force ensemble to explain the opening of base pairs and hence the denaturation of molecules in the presence of defects.

Helicase Processivity and Not the Unwinding Velocity Exhibits Universal Increase with Force

Helicases, involved in a number of cellular functions, are motors that translocate along single-stranded nucleic acid and couple the motion to unwinding double-strands of a duplex nucleic acid. The junction between double- and single-strands creates a barrier to the movement of the helicase, which can be manipulated in vitro by applying mechanical forces directly on the nucleic acid strands. Single-molecule experiments have demonstrated that the unwinding velocities of some helicases increase dramatically with increase in the external force, while others show little response. In contrast, the unwinding processivity always increases when the force increases. The differing responses of the unwinding velocity and processivity to force have lacked explanation. By generalizing a previous model of processive unwinding by helicases, we provide a unified framework for understanding the dependence of velocity and processivity on force and the nucleic acid sequence. We predict that the sensitivity of unwinding processivity to external force is a universal feature that should be observed in all helicases. Our prediction is illustrated using T7 and NS3 helicases as case studies. Interestingly, the increase in unwinding processivity with force depends on whether the helicase forces basepair opening by direct interaction or if such a disruption occurs spontaneously due to thermal fluctuations. Based on the theoretical results, we propose that proteins like single-strand binding proteins associated with helicases in the replisome may have coevolved with helicases to increase the unwinding processivity even if the velocity remains unaffected.

Entropy-driven denaturation and bubble nucleation in DNA melting

The conformational properties of a DNA molecule when mapped onto a Heisenberg spin system denaturation transition can be formulated in terms of quantum phase transition induced by a quench where the temperature effect is incorporated in the quench time. Here torsion takes on the role of the external field. The denaturation transition occurs when the entanglement entropy of the spin system vanishes. As the critical region corresponds to a two-limit behaviour the entanglement entropy gradually decreases with the gradual increase in the fraction of open base pairs and when the entropy vanishes the two strands are separated. The sequence heterogeneity interplays with the entropy effects and we have the onset of denaturation bubbles. We have studied the melting profiles for different sequence-specific DNA molecules and the results are found to be in excellent agreement with experiment.

Differential stabilities and sequence-dependent base pair opening dynamics of Watson-Crick base pairs with 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxylcytosine.

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson–Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5′-CG-3′ sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5′-T8X9G10-3′ sequence of the DDD, were compared. The presence of 5caC at the X9 base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A5:T8, whereas 5caC did not. At the oxidized base pair G4:X9, 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C3:G10. No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G4:X9; each favored Watson–Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N4 exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.

Correlating Drug Binding Affinities with Base Pair Opening Rates in DNA

The cyclic AMP responsive element (Cre) is a highly conserved stretch of DNA that is involved in the activation of gene transcription. Our group has studied the drug binding of a fluorescent derivative of a DNA intercalating anti-cancer drug, 7-amino actinomycin D (7-AMD), to the Cre sequence in different sequence contexts. We initially analyzed the DNA backbone conformation of Cre samples with varying flanking sequences and correlated these values to the binding affinities of 7-AMD. These studies revealed several anomalies that suggest that the conformation described by BI/BII content of the DNA backbone is, at most, only partially responsible for the 7-AMD binding affinity to the Cre sequence. This result is not surprising as 7-AMD has a conjugated ring structure in addition to its peptidyl side chains which interact with the backbone. As a result, we began studying DNA base pair opening rates and correlating these with 7-AMD binding affinities to account for the intercalation of 7-AMD into the Cre sequence. DNA base pair opening rates were determined by tracking imino proton exchange via NMR spectroscopy in the presence of a varying concentration of base catalyst. In this study, both two-dimensional NOESY and one-dimensional H1 NMR spectroscopy are used to track the change in line widths of the imino protons of the central Cre binding site for five sequences with varying flanking sequences. Trends in opening rates reveal that sequences with strong 7-AMD binding feature slower base pair opening dynamics than sequences with weaker 7-AMD binding. These results suggest that local base stacking is important for 7-AMD binding, a hypothesis we are currently investigating with UV spectroscopy.

NMR analysis of base-pair opening kinetics in DNA.

Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base-pair opening and closing kinetics of individual double-stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state-of-the art techniques and NMR instrumentation, including cryoprobes, is discussed.

Effect O6-guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations.

Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O(6)-alkylguanin-DNA-Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O(6)-alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O(6)-methyl-guanine (O(6)-MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O(6)-MeG:C or O(6)-MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O(6)-MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O(6)-MeG:C or O(6)-MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes.

Small-angle scattering as a tool to study the thermal denaturation of DNA

DNA thermal denaturation is the breaking of the base pairs, leading to a splitting of the two strands of the double helix. While it is easy to measure the fraction of open base pairs vs. temperature, determining the fraction of fully open molecules is much harder. Previously, the simultaneous recording of f and p could only be achieved for special sequences. We show that small-angle scattering of X-rays or neutrons allows the measurement of p for any sequence. We illustrate the method with a SAXS investigation of two sequences designed to exhibit different melting profiles and compare the SAXS data with nano-calorimetric measurements of the melting curve.

Triple helical DNA in a duplex context and base pair opening.

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.

Inhomogeneities and nonlinear dynamics of a helical DNA interacting with a RNA-polymerase

We have numerically investigated the effects of helicity and inhomogeneities on DNA base pairs opening. The inhomogeneities are due to the site-dependent stacking and hydrogen bonding energies in DNA and protein molecules. We have considered a situation in which the active site of the RNA-polymerase molecule binds onto the promoter site of the spin-like model of the DNA molecule at the physiological temperature and creates a bubble. During the study, we have found that the helical coupling has to be very weak compared to intra-strand coupling in the real DNA molecule. Results show that inhomogeneities do not affect the general pattern of base pair opening, even as the periodic inhomogeneity introduces a train of periodic oscillations on the tail of the bubble; while the height of the bubble is an increasing function of the helical coupling parameter. The basic properties of breather-like modes, obtained here by taking into account helical structure and inhomogeneities, are essential for DNA functioning since such breathing-like modes are considered to be much better candidates for the nonlinear modes responsible for a locally open state where biological functioning takes place.

Unfolding kinetics of periodic DNA hairpins

DNA hairpin molecules with periodic base sequences can be expected to exhibit a regular coarse-grained free energy landscape (FEL) as a function of the number of open base pairs and applied mechanical force. Using a commonly employed model, we first analyze for which types of sequences a particularly simple landscape structure is predicted, where forward and backward energy barriers between partly unfolded states are decreasing linearly with force. Stochastic unfolding trajectories for such molecules with simple FEL are subsequently generated by kinetic Monte Carlo simulations. Introducing probabilities that can be sampled from these trajectories, it is shown how the parameters characterizing the FEL can be estimated. Already 300 trajectories, as typically generated in experiments, provide faithful results for the FEL parameters.

Two steps forward, one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5'-3' polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein-DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).

Base Pair Opening in a Deoxynucleotide Duplex Containing a cis-syn Thymine Cyclobutane Dimer Lesion

The cis-syn thymine cyclobutane dimer is a DNA photoproduct implicated in skin cancer. We compared the stability of individual base pairs in thymine dimer-containing duplexes to undamaged parent 10-mer duplexes. UV melting thermodynamic measurements, CD spectroscopy, and 2D NOESY NMR spectroscopy confirm that the thymine dimer lesion is locally and moderately destabilizing within an overall B-form duplex conformation. We measured the rates of exchange of individual imino protons by NMR using magnetization transfer from water and determined the equilibrium constant for the opening of each base pair K(op). In the normal duplex K(op) decreases from the frayed ends of the duplex toward the center, such that the central TA pair is the most stable with a K(op) of 8 × 10⁻⁷. In contrast, base pair opening at the 5'T of the thymine dimer is facile. The 5'T of the dimer has the largest equilibrium constant (K(op) = 3 × 10⁻⁴) in its duplex, considerably larger than even the frayed penultimate base pairs. Notably, base pairing by the 3'T of the dimer is much more stable than by the 5'T, indicating that the predominant opening mechanism for the thymine dimer lesion is not likely to be flipping out into solution as a single unit. The dimer asymmetrically affects the stability of the duplex in its vicinity, destabilizing base pairing on its 5' side more than on the 3' side. The striking differences in base pair opening between parent and dimer duplexes occur independently of the duplex-single strand melting transitions.

Transcription's bubble under the influence of long-range interactions and helicoidal coupling

The influence of power-low long-range interactions (LRI) and helicoidal coupling (HC) on the properties of localized solitons in a DNA molecule when a ribonucleic acid polymerase (RNAP) binds to it at the physiological temperature is analytically and numerically investigated in this paper. We have made an analogy with the Heisenberg model Hamiltonian of an anisotropic spin ladder with ferromagnetic legs and anti-ferromagnetic rung coupling. When we limit ourselves to the second-order terms in the Taylor expansion, the DNA dynamics is found to be governed by a completely integrable nonlinear Schrödinger (NLS) equation. In this case, results show that increasing the value of HC force or LRI parameter enhances the bubble height and reduces the number of base pairs which form the bubble. For the fourth-order terms in a Taylor expansion, results are closely resembling those of second-order terms, and are confirmed by numerical investigation. These results match with some experimental data and thus provide a better representation of the base pairs opening in DNA which is essential for the transcription process.

Dynamic DNA Underpins Chromosome Dynamics

A recent article by Meyer and co-workers provides a detailed description of the dynamics of base-step conformational transitions and of the first steps to the basepair opening. These results underpin the essential role that DNA dynamics place in the DNA structural transitions that accompany the active processes of transcription, replication, and recombination in bacterial and eukaryotic chromatin.

Site-Specific Stabilization of DNA by a Tethered Major Groove Amine, 7-Aminomethyl-7-deaza-2′-deoxyguanosine

A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G1A2G3A4X5C6G7C8T9C10T11C12)2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X5 imino resonance remained sharp at 55 °C. Additionally, two 5′-neighboring base pairs, A4:T9 and G3:C10, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A4:T9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A4:T9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A4:T9 base pair in the open state of the modified duplex decreased. This was also observed for the G3:C10 base pair, where αKop for the G3:C10 base pair in the modified duplex was 3.0 × 106 versus 4.1 × 106 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X5:C8 and C6:G7 base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

Twist-DNA: computing base-pair and bubble opening probabilities in genomic superhelical DNA

Local opening of the DNA double helix is required in many fundamental biological processes and is, in part, controlled by the degree of superhelicity imposed in vivo by the protein machinery. In particular, positions of superhelically destabilized regions correlate with regulatory sites along the genome. Based on a self-consistent linearization of a thermodynamic model of superhelical DNA introduced by Benham, we have developed a program that predicts the locations of these regions by efficiently computing base-pair and bubble opening probabilities in genomic DNA. The program allows visualization of results in standard genome browsers to compare DNA opening properties with other available datasets. Source codes freely available for download at http://www.cbp.ens-lyon.fr/doku.php?id=developpement:productions:logiciels:twistdna, implemented in Fortran90 and supported on any Unix-based operating system (Linux, Mac OS X).