Spectroscopic approaches for studies of site-specific DNA base and backbone 'breathing' using exciton-coupled dimer-labeled DNA.

DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in such processes. Here we review the development and application of novel spectroscopic methods and analyses - both at the ensemble and single-molecule levels - to study structural and dynamic properties of exciton-coupled cyanine and fluorescent nucleobase analogue dimer-labeled DNA constructs at key positions involved in protein-DNA complex assembly and function. The exciton-coupled dimer probes act as 'sensors' of the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probes. These methods can be used to study the mechanisms of protein binding and function at these sites.

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.

Studies of DNA 'Breathing' by Polarization-Sweep Single-Molecule Fluorescence Microscopy of Exciton-Coupled (iCy3)2 Dimer-Labeled DNA Fork Constructs.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA 'breathing') play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here, we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3)2] dimer probes. Previous work has shown that the constructs that we tested here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3)2 dimer has one symmetric (+) and one antisymmetric (-) exciton, with the respective transition dipole moments oriented perpendicular to one another. We excite single-molecule samples using a continuous-wave linearly polarized laser, with the polarization direction continuously rotated at the frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and antisymmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and the conformational interconversion dynamics of the (iCy3)2 probe. We find that at most construct positions that we examined, the (iCy3)2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome-regulatory proteins.

Studies of Local DNA Backbone Conformation and Conformational Disorder Using Site-Specific Exciton-Coupled Dimer Probe Spectroscopy.

The processes of genome expression, regulation, and repair require direct interactions between proteins and DNA at specific sites located at and near single-stranded-double-stranded DNA (ssDNA-dsDNA) junctions. Here, we review the application of recently developed spectroscopic methods and analyses that combine linear absorbance and circular dichroism spectroscopy with nonlinear 2D fluorescence spectroscopy to study the local conformations and conformational disorder of the sugar-phosphate backbones of ssDNA-dsDNA fork constructs that have been internally labeled with exciton-coupled cyanine (iCy3)2 dimer probes. With the application of these methods, the (iCy3)2 dimer can serve as a reliable probe of the mean local conformations and conformational distributions of the sugar-phosphate backbones of dsDNA at various critical positions. The results of our studies suggest a possible structural framework for understanding the roles of DNA breathing in driving the processes of protein-DNA complex assembly and function.

Temperature-dependent local conformations and conformational distributions of cyanine dimer labeled single-stranded-double-stranded DNA junctions by 2D fluorescence spectroscopy.

DNA replication and the related processes of genome expression require binding, assembly, and function of protein complexes at and near single-stranded (ss)-double-stranded (ds) DNA junctions. These central protein-DNA interactions are likely influenced by thermally induced conformational fluctuations of the DNA scaffold across an unknown distribution of functionally relevant states to provide regulatory proteins access to properly conformed DNA binding sites. Thus, characterizing the nature of conformational fluctuations and the associated structural disorder at ss-dsDNA junctions is critical for understanding the molecular mechanisms of these central biological processes. Here, we describe spectroscopic studies of model ss-dsDNA fork constructs that contain dimers of "internally labeled" cyanine (iCy3) chromophore probes that have been rigidly inserted within the sugar-phosphate backbones of the DNA strands. Our combined analyses of absorbance, circular dichroism, and two-dimensional fluorescence spectroscopy permit us to characterize the local conformational parameters and conformational distributions. We find that the DNA sugar-phosphate backbones undergo abrupt successive changes in their local conformations-initially from a right-handed and ordered DNA state to a disordered splayed-open structure and then to a disordered left-handed conformation-as the dimer probes are moved across the ss-dsDNA junction. Our results suggest that the sugar-phosphate backbones at and near ss-dsDNA junctions adopt specific position-dependent local conformations and exhibit varying extents of conformational disorder that deviate widely from the Watson-Crick structure. We suggest that some of these conformations can function as secondary-structure motifs for interaction with protein complexes that bind to and assemble at these sites.

Submillisecond Conformational Transitions of Short Single-Stranded DNA Lattices by Photon Correlation Single-Molecule Förster Resonance Energy Transfer.

Thermally driven conformational fluctuations (or "breathing") of DNA play important roles in the function and regulation of the "macromolecular machinery of genome expression." Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here, we use microsecond-resolved single-molecule Förster resonance energy transfer (smFRET) experiments to investigate the backbone fluctuations of short [oligo(dT)n] templates within DNA constructs that also serve as models for ss-dsDNA junctions. Such junctions, together with the attached ssDNA sequences, are involved in interactions with the ssDNA binding (ssb) proteins that control and integrate the functions of DNA replication complexes. We analyze these data using a chemical network model based on multiorder time-correlation functions and probability distribution functions that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs interconvert, on submillisecond time scales, between three macrostates with distinctly different end-to-end distances. These are (i) a "compact" macrostate that represents the dominant species at equilibrium; (ii) a "partially extended" macrostate that exists as minority species; and (iii) a "highly extended" macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA-associating proteins.

Dinucleotides as simple models of the base stacking-unstacking component of DNA 'breathing' mechanisms.

Regulatory protein access to the DNA duplex 'interior' depends on local DNA 'breathing' fluctuations, and the most fundamental of these are thermally-driven base stacking-unstacking interactions. The smallest DNA unit that can undergo such transitions is the dinucleotide, whose structural and dynamic properties are dominated by stacking, while the ion condensation, cooperative stacking and inter-base hydrogen-bonding present in duplex DNA are not involved. We use dApdA to study stacking-unstacking at the dinucleotide level because the fluctuations observed are likely to resemble those of larger DNA molecules, but in the absence of constraints introduced by cooperativity are likely to be more pronounced, and thus more accessible to measurement. We study these fluctuations with a combination of Molecular Dynamics simulations on the microsecond timescale and Markov State Model analyses, and validate our results by calculations of circular dichroism (CD) spectra, with results that agree well with the experimental spectra. Our analyses show that the CD spectrum of dApdA is defined by two distinct chiral conformations that correspond, respectively, to a Watson-Crick form and a hybrid form with one base in a Hoogsteen configuration. We find also that ionic structure and water orientation around dApdA play important roles in controlling its breathing fluctuations.

Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution.

In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.

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.

Measuring local conformations and conformational disorder of (Cy3)2 dimer labeled DNA fork junctions using absorbance, circular dichroism and two-dimensional fluorescence spectroscopy.

The sugar-phosphate backbone of DNA near single-stranded (ss)-double-stranded (ds) junctions likely fluctuates within a broad distribution of conformations to permit the proper binding of genome regulatory proteins that function at these sites. In this work we use absorbance, circular dichroism (CD), and two-dimensional fluorescence spectroscopy (2DFS) to study the local conformations and conformational disorder within chromophore-labeled DNA constructs. These constructs employ dimers of the fluorescent chromophore Cy3 that are site-specifically incorporated into the sugar-phosphate backbones of DNA strands at ss-ds DNA fork junctions. We show that these data can be analyzed to determine the local conformations of the (Cy3)2 dimer, and the degree of conformational disorder. Our analysis employs an essential-state Holstein-Frenkel Hamiltonian model, which takes into account the internal electronic-vibrational motions within each Cy3 chromophore, and the resonant electronic interaction that couples the two chromophores together. Our results suggest that this approach may be applied generally to understand local backbone conformation and conformational disorder at ss-ds DNA fork junctions.

Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks.

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions.

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can 'slide' on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.

Sequence-Dependent Conformational Heterogeneity and Proton-Transfer Reactivity of the Fluorescent Guanine Analogue 6-Methyl Isoxanthopterin (6-MI) in DNA.

The local conformations of individual nucleic acid bases in DNA are important components in processes fundamental to gene regulation. Fluorescent nucleic acid base analogues, which can be substituted for natural bases in DNA, can serve as useful spectroscopic probes of average local base conformation and conformational heterogeneity. Here we report excitation-emission peak shift (EES) measurements of the fluorescent guanine (G) analogue 6-methyl isoxanthoptherin (6-MI), both as a ribonucleotide monophosphate (NMP) in solution and as a site-specific substituent for G in various DNA constructs. Changes in the peak positions of the fluorescence spectra as a function of excitation energy indicate that distinct subpopulations of conformational states exist in these samples on time scales longer than the fluorescence lifetime. Our pH-dependent measurements of the 6-MI NMP in solution show that these states can be identified as protonated and deprotonated forms of the 6-MI fluorescent probe. We implement a simple two-state model, which includes four vibrationally coupled electronic levels to estimate the free energy change, the free energy of activation, and the equilibrium constant for the proton transfer reaction. These parameters vary in single-stranded and duplex DNA constructs, and also depend on the sequence context of flanking bases. Our results suggest that proton transfer in 6-MI-substituted DNA constructs is coupled to conformational heterogeneity of the probe base, and can be interpreted to suggest that Watson-Crick base pairing between 6-MI and its complementary cytosine in duplex DNA involves a "low-barrier-hydrogen-bond". These findings may be important in using the 6-MI probe to understand local base conformational fluctuations, which likely play a central role in protein-DNA and ligand-DNA 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.

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

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5'-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.