Local DNA Conformation Research Articles

Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2 dimer probes of local DNA conformations at or near functional protein binding sites.

Exciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of 'internally-labeled' (iCy3)2 dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2 dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2 dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.

Protein-DNA recognition mechanisms and specificity.

The accumulated knowledge about the structure of protein-DNA complexes allowed us to understand the mechanisms of protein-DNA recognition and searching for a specific site on DNA. Obviously, the mechanism of specific DNA recognition by a protein must satisfy two requirements. First, the probability of incorrect binding should be very small. Second, the time to find the "correct" binding site should not be too long. If we assume that protein recognition of a precise site on DNA occurs at some distance from DNA and calculate global minima, we can avoid local minima at short distances. The only long-range interaction is the interaction of charges. The location of charges on DNA in three-dimensional space depends on the local conformation of DNA and thus reflects the DNA sequence and sets the spatial pattern for recognition. Various factors such as counter ion concentration, ionic strength, and pH can affect protein recognition of DNA. Nowadays, the theory of long-range interactions makes it possible to calculate the best mutual spatial arrangement of protein and DNA molecules by charged groups and avoid misplaced binding.

Investigating local DNA conformational trapping dynamics during DNA polymerase holoenzyme assembly and exonuclease proof-reading activity.

The sugar-phosphate backbones of DNA continuously undergo thermally-activated conformational fluctuations at equilibrium, which directly affect base-pair unstacking and hydrogen bond breaking. The influence of these motions on biomolecular function are particularly important at single-stranded (ss)—double-stranded (ds) DNA forks and primer-template (p/t) junctions. At these junctions, non-canonical structures that deviate from the Watson-Crick B-form DNA duplex may serve as recognition sites for protein binding, and the kinetics of these interactions likely play a central regulatory role for fundamental biological processes such as DNA replication and repair. The DNA polymerase/sliding clamp holoenzyme is the central protein complex that carries out the process of complementary nucleotide addition to nascent ssDNA template strands initiated at p/t junctions. In addition to playing a role in protein assembly, local DNA conformations resulting from nucleotide misincorporation regulate the exonuclease proof-reading activity of DNA polymerase. To study the details of these processive local DNA conformational dynamics, our group has developed a molecular probing method that relies on the rigid insertion of two fluorescent cyanine (Cy3) probes at opposite positions within the sugar-phosphate backbones of complementary DNA strands. Electrostatic coupling between probes results in delocalized optical properties that sensitively depend on relative probe orientations. Utilizing a series of complementary ensemble-based (i.e., fluorescence, absorbance, circular dichroism) and single-molecule spectroscopic methods, we study the local DNA backbone conformational dynamics as they relate to protein binding and activity. In this presentation, I will discuss recent studies of DNA polymerase holoenzyme assembly and exonuclease proofreading activity at p/t junctions. Specifically, results from microsecond-resolved polarization-sweep single-molecule spectroscopy (PS-SMS) demonstrate how protein-dependent conformational trapping of otherwise sparsely populated transient DNA local conformations is involved in processive holoenzyme formation.

Effect of a U:G mispair on the water around DNA

DNA repair proteins are able to discriminate DNA lesions among an abundance of intact DNA with high selectivity. To investigate detectable characteristics of one specific lesion, we compare statistical results from molecular dynamics simulations of two different DNA in water, one with an intact C:G pair and one that contains a U:G mispair, and perform a comparative analysis of the water dynamics around the two. Our data show that in addition to the local DNA conformation, also the surrounding water shell exhibits significant differences that may help mispair discrimination. The chemical groups which account for a U:G mispair to exhibit a wobble conformation instead of the ‘proper’ Watson-Crick pairing of a C:G pair, that is an oxygen atom (in uracil) instead of an amino group (in cytosine), also order the water molecules around the bases such that they act predominantly as hydrogen-bond donor or acceptor to the uracil or cytosine base, respectively. These changes in water conformation stretch into the second solvation shell, which may be exploited by repair enzymes to achieve lesion detection with high efficiency.

Investigating the role of local DNA conformational dynamics in processive DNA polymerase holoenzyme-primer-template DNA complex formation

Single-strand-double-strand (ss-ds) DNA junctions play important roles in protein recognition and binding interactions in DNA replication and repair. The functionality of ss-dsDNA junctions is derived, at least in part, from intrinsic disorder at these sites that results in non-canonical local DNA conformations and conformational dynamics. Primer/template (P/T) DNA junctions serve as sites for the multistep assembly of the DNA polymerase/sliding clamp holoenzyme that drives DNA replication and proof-reading.

Determining Local Conformations and Conformational Disorder at and Near Single-Stranded (Ss) and Double-Stranded (Ds) DNA Forks and Junctions

DNA functions as a stable repository for heritable information across generations. However, the structure of DNA within the cell must be dynamic, allowing for thermally induced fluctuations to facilitate the recognition and assembly of functional protein-DNA complexes. The local conformations of the sugar-phosphate backbones near the replication fork junction are likely recognized by protein components that form a stable helicase-primase sub-assembly during DNA replication. Moreover, the presence of local backbone disorder within duplex and ss-ds DNA junctions indicates a distribution of local backbone conformations that could, for example, facilitate kinetic competition between distinct protein regulatory factors. The exploration of local DNA conformations can be examined by site-specifically inserting cyanine (Cy3) dimers within the sugar-phosphate backbones of DNA constructs. Spectroscopic characterization of these Cy3 dimer labeled DNA constructs can provide detailed, site-specific information about the local conformations and disorder of the sugar-phosphate backbones at the Cy3 labeling site positions. By fitting a theoretical model to experimental absorbance and circular dichroism (CD) spectra, we determined the ensemble average conformations (relative orientation and distances) of Cy3 dimer probes within the DNA constructs as a function of insertion site position and temperature. We compared the results of our analyses using increasingly complex models of exciton coupling between individual Cy3 labeling sites. To investigate local conformational disorder of the sugar-phosphate backbones as a function of temperature and proximity to protein binding sites, we used two-dimensional fluorescence spectroscopy (2DFS), which allowed us to characterize local conformational disorder at the Cy3 labeling sites. The presence of local disorder at ss-ds DNA junctions suggests that these sites undergo rapid interconversion between different conformations. Single-molecule polarization-sweep experiments were used to obtain detailed molecular information about the mechanisms of these conformational transitions.

Connecting Structure with Kinetics of Single-Stranded DNA Fluctuations that Provide Potential Binding Sites for Replication Proteins

The local conformations of DNA at and near replication forks and primer-template (p/t) junctions likely regulate the non-sequence-specific binding of replication proteins. Thermal fluctuations can drive the exploration of local DNA conformations, thereby providing opportunities for proteins to stably bind to transiently formed secondary structure motifs. In this work, we apply microsecond-resolved single-molecule Förster resonance energy transfer (sm FRET) experiments to study the equilibrium distribution and conformational transitions of the end-to-end distances of an oligo-deoxythymidine chain (dT)15 that serves as the single-stranded (ss) region of a Cy3-Cy5 labeled p/t DNA construct. By comparing our experimental data to the results of a kinetic network model, we determined that the system exists as three distinct macrostates with different end-to-end extensions, and that transitions between these macrostates occur over time scales ranging from ∼ 10 microseconds to ∼ 100 milliseconds. In addition, we measured the relative stabilities and transition barrier heights that characterize the free energy landscape of the (dT)15 p/t DNA system. The magnitudes of the activation barriers between macrostates of varying chain extension likely depend on the rates of rearrangement of the various populations of base stacked segments within the single-stranded portion of the p/t DNA construct. In ongoing work, we are investigating the physical nature of the transition barriers between macrostates in terms of local base-stacking interactions, using a combination of Monte Carlo simulation and spectroscopy of fluorescent base analogue substituted DNA constructs. Certain conformations within these macrostates are likely to represent targets for the binding of proteins, such as the single-stranded DNA binding protein (gp32) of bacteriophage T4, to ssDNA templates in the assembly and function of the replication complex.

Kinetics of Photoinduced Reactions at the Single-Molecule Level: The KACB Method.

The kinetics of photoinduced reactions can be approached by laser flash photolysis techniques. Although such techniques allow for a detailed understanding of the important photophysics of molecules, they normally require a substantial amount of sample for measurements (>1 nmol), and thus, they are difficult to apply to analytical and diagnostic applications. The photophysics of a fluorescent molecule can be accessed by monitoring the kinetics of the fluctuation of fluorescence, which is called blinking. Blinking is a phenomenon that can be monitored only if molecules are observed at the single-molecule level. In bulk solution, blinking kinetics can be measured by using fluorescence correlation spectroscopy (FCS), which normally requires more than 105 times less sample than that required for laser flash photolysis. Blinking is controlled to extract fruitful microenvironmental information around a fluorescent molecule, by using a method named kinetic analysis based on the control of fluorescence blinking (KACB). This Concept highlights the adaption of the KACB method to investigate the local conformation of DNA with less than 1 pmol of DNA sample.

2D Fluorescence Spectroscopy is used to Probe Local Conformations and Conformational Disorder of the Sugar-Phosphate Backbones of DNA at and Near DNA Replication Fork Junctions

DNA replication requires the binding of genome-regulatory proteins to non-sequence-specific positions near single-stranded (ss) - double-stranded (ds) DNA junctions. The local conformation of DNA near ss-dsDNA junctions must undergo fluctuations within an unknown distribution of functionally relevant states to permit the proper binding of replication proteins. As a consequence, the nature and extent of conformational disorder at DNA junctions may be critical for controlling the molecular mechanisms of this binding. DNA constructs that contain dimers of Cy3 chromophore probes rigidly incorporated into the sugar-phosphate backbones at defined positions near ss-dsDNA fork junctions have been studied using absorption, circular dichroism (CD) and two-dimensional fluorescence spectroscopic (2DFS) techniques to determine local backbone conformations and the degree of disorder experienced by these dimer probes. Analysis of these data provides insight into position- and temperature-dependent conformational heterogeneity at various sites within these DNA constructs, and suggests that quantifiably different energetic barriers to conformational exploration exist at the ss-ds junction when compared to DNA base pairs located deeper within the duplex DNA. We have employed an optimization routine to fit linear and nonlinear spectra in order to characterize structural and disorder parameters associated with conformations at and near ss-dsDNA junctions that are stabilized by the binding of relevant proteins, as well as to examine structural distortions induced by DNA-associated small molecules and ions.

Local DNA Base Conformations and Ligand Intercalation in DNA Constructs Containing Optical Probes

Understanding local conformations of DNA at the level of individual nucleic acid bases and base pairs is important for elucidating molecular processes that depend on DNA sequence. Here, we apply linear absorption and circular dichroism measurements to the study of local DNA conformations, using the guanine base analog 6-methyl isoxanthopterin (6-MI) as a structural probe. We show that the spectroscopic properties of this probe can provide detailed information about the average local base and basepair conformations as a function of the surrounding DNA sequence. Based on these results we apply a simple theoretical model to calculate the circular dichroism spectra of 6-MI-substituted DNA constructs and show that our model can be used to extract information about how the local conformations of the 6-MI probe are influenced by the local base or basepair environment. We also use this probe to examine the pathway for the insertion (intercalation) of a tethered acridine ligand (9-amino-6-chloro methoxyacridine) into duplex DNA. We show that this model intercalator interacts with duplex DNA by a “displacement insertion intercalation” mechanism, whereby the acridine moiety is inserted into the DNA structure and displaces the base located opposite its attachment site. These findings suggest that site-specifically positioned base analog probes can be used to characterize the molecular and structural details of binding ligand effects on local base stacking and unstacking reactions in single- and double-stranded DNA and thus may help to define the molecular mechanisms of DNA-protein interactions that involve the site-specific intercalation of aromatic amino acid side chains into genomic DNA.

Electron Transfer in Spacer-Free DNA Duplexes Tethered to Gold via dA10 Tags.

Electrical properties of DNA critically depend on the way DNA molecules are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, we show that the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to the hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage ΓDNA (with a linear regression limit of 670 s-1 at ΓDNA → 0), being 2.7-fold enhanced compared to phosphorothioated dA5* tethering (6-fold for the C6-alkanethiol linker representing an additional ET barrier). X-ray photoelectron spectroscopy evidenced dA10 binding to the Au surface via the purine N, whereas dA5* predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter apparently induces the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing the ET rates through the duplex. Thus, a spacer-free DNA coupling to electrodes via dA10 tags thus allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive, synthetic linkers.

Does Cytosine Methylation Stabilize the BI Substate of DNA?

The local conformation of DNA plays a critical role in the recognition-and-binding process of transcription factors. In particular, the most common form of the DNA double helix, B-DNA, exists as a conformational equilibrium between BI and BII substates that are associated with changes in major and minor groove dimensions. It has been hypothesized that cytosine methylation, the most common epigenetic modification and one that is often found in so-called CpG islands, modulates transcription factor binding affinity by altering the BI-BII equilibrium in the neighborhood of methylation sites. Indeed, previous molecular dynamics studies have suggested that cytosine methylation leads to a uniform stabilization of the BI substate in CpG islands. In this work we first benchmark the ability of the latest Amber DNA force fields and a variety of three-point water models to reproduce BI-BII equilibrium in the Dickerson dodecamer and reduce terminal base pair fraying. Using the best-performing combinations, we then re-examine the conformational effects of cytosine methylation in a prototypical CpG island, (GC)5. We find that the BI substate is stabilized for GpC steps in such sequences, but that the BII substate is stabilized for the CpG steps. More interestingly, the Amber ff99bsc0 DNA force field used in previous studies yields opposite conclusions. These findings highlight the need for continued fixed-charge force field development for nucleic acids, as well as solution state experimental datasets involving epigenetic modification that can serve as benchmarks for simulation accuracy.

Investigating the effect of charged amino acids on DNA conformation in EcoRI–DNA complex: a molecular dynamics simulation study

Sequence-specific binding of proteins to DNA is essential for almost all the cellular processes like transcription, translation, replication, etc. One among the various mechanisms that has been identified so far that contributes to the specificity in protein–DNA interaction is the DNA conformational change. Electrostatic neutralization of the phosphate groups by the positively charged amino acid residues in proteins is thought to bring about such conformational changes in DNA. Here, we employ molecular dynamics simulations to examine the effect of charge on amino acids Lys113, Arg145, and Asp91 which are attached to the scissile phosphate on the conformation of DNA in EcoRI–DNA complex. The results indicate that the charge of these amino acids is essential for maintaining the local conformation of DNA in the EcoRI-bound form. Interestingly, we observe that the positively charged amino acids Lys113 and Arg145 have a long-range influence on the DNA conformation, whereas the negatively charged amino acid Asp91 has only a localized effect on the DNA conformation. The charge on the amino acids also alters the collective dynamics of EcoRI. Collectively, the results shed light on the diversity of the effect of charges on DNA conformation as well as on protein dynamics.

Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy.

This article reviews atomic force microscopy (AFM) studies of DNA structure and dynamics and protein-DNA complexes, including recent advances in the visualization of protein-DNA complexes with the use of cutting-edge, high-speed AFM. Special emphasis is given to direct nanoscale visualization of dynamics of protein-DNA complexes. In the area of DNA structure and dynamics, structural studies of local non-B conformations of DNA and the interplay of local and global DNA conformations are reviewed. The application of time-lapse AFM nanoscale imaging of DNA dynamics is illustrated by studies of Holliday junction branch migration. Structure and dynamics of protein-DNA interactions include problems related to site-specific DNA recombination, DNA replication, and DNA mismatch repair. Studies involving the structure and dynamics of chromatin are also described.

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

Local Compression Properties of Double-Stranded DNA Based on a Dynamic Simulation

Using a simple sphere-tip compression system, the local radial mechanical properties of DNA are systematically studied by changing the tip size. When the tip size decreases, the radial compression elastic properties under external loads become sensitive to the tip size and the local DNA conformation. A sudden force break appears in the compression-force curve when the tip size is less than or equal to 12 nm in diameter. The analysis of the hydrogen bonds and the base stacking interaction shows that a local unwinding process occurs. During the compression process, firstly the hydrogen bonds between complement base pairs are broken. With the compression accumulating, the local backbones in the compression center are unwound from the double helix conformation to a kind of parallel conformation. This local unwinding behavior deduced by external loads is helpful to understand the biological process and is important to DNA-based nanomechanical devices.

Spectroscopic analysis of the interaction of lomustine with calf thymus DNA

Investigation of drug–DNA interaction is important for understanding the drug action at molecular level and for designing specific DNA targeted drug. Lomustine (CCNU=1-[2-chloroethyl]-3-cyclohexyl-1-nitroso-urea) is an alkylating antineoplastic nitrosourea derivative, used to treat different types of cancer. In the present study, conformational and structural effects of lomustine on DNA are investigated using different spectroscopic approaches. Different drug/DNA molar ratios are analyzed to determine the binding sites and binding mode of lomustine with DNA. Fourier transform infrared spectroscopic (FTIR) results suggest binding of lomustine with nitrogenous bases guanine and cytosine along with weak interaction to the sugar-phosphate backbone of DNA. Circular dichroism (CD) spectroscopic results show perturbation in the local conformation of DNA upon binding of lomustine with DNA helix. These local conformational changes may act as recognition site for alkylating enzymes that further causes alkylation of DNA. Spectroscopic results confirm the formation of an intermediate stage of DNA that occurs during the transition of B-conformation into A-conformation.

Automatic workflow for the classification of local DNA conformations

BackgroundA growing number of crystal and NMR structures reveals a considerable structural polymorphism of DNA architecture going well beyond the usual image of a double helical molecule. DNA is highly variable with dinucleotide steps exhibiting a substantial flexibility in a sequence-dependent manner. An analysis of the conformational space of the DNA backbone and the enhancement of our understanding of the conformational dependencies in DNA are therefore important for full comprehension of DNA structural polymorphism.ResultsA detailed classification of local DNA conformations based on the technique of Fourier averaging was published in our previous work. However, this procedure requires a considerable amount of manual work. To overcome this limitation we developed an automatic classification method consisting of the combination of supervised and unsupervised approaches. A proposed workflow is composed of k-NN method followed by a non-hierarchical single-pass clustering algorithm. We applied this workflow to analyze 816 X-ray and 664 NMR DNA structures released till February 2013. We identified and annotated six new conformers, and we assigned four of these conformers to two structurally important DNA families: guanine quadruplexes and Holliday (four-way) junctions. We also compared populations of the assigned conformers in the dataset of X-ray and NMR structures.ConclusionsIn the present work we developed a machine learning workflow for the automatic classification of dinucleotide conformations. Dinucleotides with unassigned conformations can be either classified into one of already known 24 classes or they can be flagged as unclassifiable. The proposed machine learning workflow permits identification of new classes among so far unclassifiable data, and we identified and annotated six new conformations in the X-ray structures released since our previous analysis. The results illustrate the utility of machine learning approaches in the classification of local DNA conformations.

An insulin-like growth factor-II intronic variant affects local DNA conformation and ovarian cancer survival

Insulin-like growth factor-II (IGF-II) may be a prognostic marker in ovarian cancer, and its intronic single nucleotide polymorphism (SNP) rs4320932 has been associated with risk of the disease. We determined whether rs4320932 is associated with IGF-II expression and patient survival in ovarian cancer, and explored whether the SNP variation affects DNA conformation both in the absence of and presence of carboplatin. IGF-II genotype (rs4320932) and phenotype were analyzed in 212 primary invasive epithelial ovarian cancer tissue samples with Taqman® SNP genotyping assays, quantitative reverse transcription-polymerase chain reaction and commercial enzyme-linked immunosorbent assay. DNA conformation was evaluated by circular dichroism (CD) spectra. Kaplan-Meier survival curves and Cox proportional hazard regression models were used to analyze the SNP associations with patient survival. The C allele of rs4320932, previously associated with decreased risk of ovarian cancer development, was here associated with significantly elevated risks of relapse (Ptrend = 0.0002) and death (Ptrend = 0.0006), remaining significant in multivariate analyses. The adjusted hazard ratios were 3.05 (95% confidence interval [CI]: 1.47-6.37) for relapse and 3.28 (95% CI: 1.64-6.57) for death, respectively. The variant was also significantly associated with chemotherapy response, but not with other clinicopathologic variables or with IGF-II expression. DNA with genotypes TT and CC had distinct CD spectra in both the absence of and presence of carboplatin. These findings suggest that the intronic SNP rs4320932 affects patient survival and chemotherapy response via alteration of DNA conformation, but not through regulation of IGF-II expression. This novel finding may have implications in individualized medicine for the design of specific molecules targeting DNA of specific conformations.

Multiple RNA Polymerase Transactions on Single DNA Templates

We are studying the interactions of more than one RNA polymerase (RNAP) operating on single linear double-stranded DNA templates using imaging by atomic force microscopy (AFM) [1,2]. These templates include a short polynucleotide sequence of A, C, G or T forming a single-stranded end loop to discriminate polarity of the DNA molecules in the AFM [3]. Model templates include convergently and tandemly-aligned promoters for bacterial E. Coli RNAP.Measurement of the positions of the RNAP on the DNA before and after firing from the promoters allow us infer outcomes of “collision” events. The end-labelling ssDNA loop not only acts as a fiducial marker but also captures RNAPs that have fired from promoters directed towards and closest to the loop. This enables us to quantify different outcomes of these events between more than one RNAP operating in a convergent or tandem configuration. Interestingly, RNAPs which approach each other often stall before close contact is made, implying that transient DNA super-coiling between RNAP mediates their interactions. Furthermore, the distribution of outcomes for head-on (convergent transcription) and rear-end (tandem transcription) collision events are similar, suggesting that long range interactions mediated through the local DNA conformation serve to regulate RNAP activity and positioning on the templates. These experiments inform the regulation of genetic expression at the molecular level, including acting as simple models for nested genes.[1] Billingsley D.J., Bonass W.A, Crampton N., Kirkham J. and Thomson N.H. (2012) Physical Biology 9 021001.[2] Crampton N., Bonass W.A., Kirkham J., Rivetti C. and Thomson N.H. (2006) Nucleic Acids Research 34 (19) 5416-5425.[3] Billingsley D.J., Crampton N., Kirkham J., Thomson N.H., Bonass W.A (2012) Nucleic Acids Research 40 (13) e99.