Towards single-molecule DNA sequencing: assays with low nonspecific adsorption

New DNA sequencing techniques are currently being developed using single-molecule fluorescence-based detection of enzymatic double-strand synthesis. Such application requires surface architectures on which single-stranded templates can be immobilized. A further important attribute is a very low tendency to attract fluorescently labeled bases nonspecifically. On this account, the adsorption behaviour of Cy5-dNTPs on a variety of surface coatings was studied by performing real-time measurements of the DNA synthesis using a supercritical angle fluorescence biosensor. It is demonstrated that polyacrylic acid coatings are an excellent choice to minimize the nonspecific binding of the bases.     

Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation

In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the subsequent binding of polymerase to the loaded clamp. Recently published evidence suggests that holoenzyme assembly in the T4 replisome may take place via more than one pathway [Zhuang, Z., Berdis, A. J., and Benkovic, S. J. (2006) Biochemistry 45, 7976-7989]. To demonstrate unequivocally whether there are multiple pathways leading to the formation of a productive holoenzyme, single-molecule fluorescence microscopy has been used to study the potential clamp loading and holoenzyme assembly pathways on a single-molecule DNA substrate. The results obtained reveal four pathways that foster the formation of a functional holoenzyme on DNA: (1) clamp loader-clamp complex binding to DNA followed by polymerase, (2) clamp loader binding to DNA followed by clamp and then polymerase, (3) clamp binding to DNA followed by clamp loader and then polymerase, and (4) polymerase binding to DNA followed by the clamp loader-clamp complex. In all cases, MgATP is required. The possible physiological significance of the various assembly pathways is discussed in the context of replication initiation and lagging strand synthesis during various stages of T4 phage replication.     

Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer

Many studies of specific protein-nucleic acid binding use short oligonucleotides or restriction fragments, in part to minimize the potential for nonspecific binding of the protein. However, when the specificity ratio is low, multiple nonspecifically bound proteins may occupy the region of DNA corresponding to one specific site; this situation was encountered in our recent calorimetric study of binding of integration host factor (IHF) protein to its specific 34-bp H' DNA site. Here, beginning from the analytical McGhee and von Hippel infinite-lattice nonspecific binding isotherm, we derive a novel analytic isotherm for nonspecific binding of a ligand to a finite lattice. This isotherm is an excellent approximation to the exact factorial-based Epstein finite lattice isotherm even for short lattices and therefore is of great practical significance for analysis of experimental data and for analytic theory. Using this isotherm, we develop an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecific binding sites. Analysis of calorimetric data for IHF-H' DNA binding using this treatment yields enthalpies and binding constants for both specific and nonspecific binding and the nonspecific site size. This novel analysis demonstrates the potential contribution of nonspecific binding to the observed thermodynamics of specific binding, even with very short DNA oligomers, and the need for reverse (constant protein) titrations or titrations with nonspecific DNA to resolve specific and nonspecific contributions. The competition treatment is useful in analyzing low-specificity systems, including those where specificity is weakened by mutations or the absence of specificity factors.     

Function and assembly of the bacteriophage T4 DNA replication complex: interactions of the T4 polymerase with various model DNA constructs

Complexes formed between DNA polymerase and genomic DNA at the replication fork are key elements of the replication machinery. We used sedimentation velocity, fluorescence anisotropy, and surface plasmon resonance to measure the binding interactions between bacteriophage T4 DNA polymerase (gp43) and various model DNA constructs. These results provide quantitative insight into how this replication polymerase performs template-directed 5' --> 3' DNA synthesis and how this function is coordinated with the activities of the other proteins of the replication complex. We find that short (single- and double-stranded) DNA molecules bind a single gp43 polymerase in a nonspecific (overlap) binding mode with moderate affinity (Kd approximately 150 nm) and a binding site size of approximately 10 nucleotides for single-stranded DNA and approximately 13 bp for double-stranded DNA. In contrast, gp43 binds in a site-specific (nonoverlap) mode and significantly more tightly (Kd approximately 5 nm) to DNA constructs carrying a primer-template junction, with the polymerase covering approximately 5 nucleotides downstream and approximately 6-7 bp upstream of the 3'-primer terminus. The rate of this specific binding interaction is close to diffusion-controlled. The affinity of gp43 for the primer-template junction is modulated specifically by dNTP substrates, with the next "correct" dNTP strengthening the interaction and an incorrect dNTP weakening the observed binding. These results are discussed in terms of the individual steps of the polymerase-catalyzed single nucleotide addition cycle and the replication complex assembly process. We suggest that changes in the kinetics and thermodynamics of these steps by auxiliary replication proteins constitute a basic mechanism for protein coupling within the replication complex.     

Effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I

In the presence of the Nt.BspD6I nicking endonuclease DNA polymerase Bst stimulates intensive template/primer-independent DNA synthesis. Template/primer-independent DNA synthesis could be the reason for appearing nonspecific DNA products in many DNA amplification reactions particularly in the reactions with using nicking endonucleases. Search of the modes for inhibition template/primer-independent DNA synthesis becomes an urgent task because of broadening the DNA amplification methods with using nicking endonucleases. We report here that the E. coli single-stranded DNA binding protein has no effect on the template/primer-independent DNA synthesis. In the absence of the nicking endonuclease the single-stranded DNA binding protein encoded by bacteriophage T4 gene 32 completely inhibits template/primer-independent DNA synthesis. This protein does not inhibit synthesis of specific DNA product in the presence of nicking endonuclease but remarkably decreases the amount of nonspecific products.     

Isolation of viral specific RNA from SV40 infected cells by viral DNA chemically linked to a cellulose matrix.

SV40 DNA fragments chemically attached to neutral cellulose powder with a water-soluble carbodiimide have been used to isolate late lytic viral specific RNA from virus infected cells. Exhaustive hybridization to SV40 DNA reveals that virtually all of the isolated RNA molecules contain SV40 specific sequences. Comparison with SV40 cRNA prepared with purified Escherichia coli RNA polymerase and a SV40 DNA I template suggests that the purity of the isolated SV40 specific RNA is very close to 100%. The background level for the nonspecific binding of RNA to a purified cellulose matrix is very low. Retention of nonspecific RNA by SV40 DNA-cellulose is only 1.5% of the viral specific RNA isolated under saturating conditions for the column. Sedimentation in neutral sucrose suggests that the major 16S viral specific RNA has been isolated largely intact.     

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We present the first structural model of a bacteriophage T7 DNA helicase-DNA polymerase complex using a combination of small angle x-ray scattering, single-molecule, and biochemical methods. We propose that the protein-protein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. DNA primase facilitates binding of DNA helicase to ssDNA and contributes to formation of the DNA helicase-DNA polymerase complex by stabilizing DNA helicase.     

Carcinogenic adducts induce distinct DNA polymerase binding orientations

DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism.     

Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions

The difference boundary sedimentation velocity technique of Schachman and co-workers is demonstrated to be applicalbe to the measurement of binding constants (Kobsd) in the range 10(2)-10(5) M(-1) for the nonspecific interactions of proteins with DNA. The difference technique can reproducibly detect a 2% change in the sedimentation coefficient of the DNA upon binding ligands, corresponding to average extents of association as low as 10 molecules of protein (in the cases of Escherichia coli lac repressor and E. coli RNA polymerase) per molecule of bacteriophage T7 DNA. At these low binding densities, it is plausible to assume that the primary effect of ligand binding is on the buoyant mass of the complex and not on the frictional coefficient of the flexible DNA coil. Binding constants calculated by using this assumption agree well with literature values for the nonspecific interactions of RNase and lac repressor proteins with double-stranded DNA. Advantages of the method are that it is relatively rapid, requires the optical detection of the DNA only, and can be performed on small amounts of sample. The method appears useful for surveying (to an accuracy of +/-50% in Kobsd or +/-10% in log Kobsd) the effects of solution variables on Kobsd of protein-DNA interactions. Applications of the method to the nonspecific interactions of RNA polymerase core and holoenzymes with T7 DNA are discussed.     

Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.     

Promoter search by Escherichia coli RNA polymerase on a circular DNA template

Using the rapid-mixing/photocross-linking technique developed in our laboratory, we have investigated the kinetics of interaction between Escherichia coli RNA polymerase and pAR1319, a recombinant plasmid DNA containing the bacteriophage T7 A2 early promoter. By monitoring the time-dependent density of bound RNA polymerase along the relaxed circular DNA molecule using this technique, we have been able to demonstrate kinetic evidence for linear diffusion of RNA polymerase along DNA in a different system from that previously described (Park, C. S., Hillel, Z., and Wu, C.-W. (1982) J. Biol. Chem. 251, 6950-6956). The nonspecific association rate constant kon was measured to be 7.7 x 10(4) M-1 s-1 at a DNA chain concentration of 22.4 nM. By taking advantage of the fact that rapid mixing displaces bound protein molecules from DNA, but leaves them within the domain of the DNA, the rate of intradomain binding of RNA polymerase to pAR1319 DNA was determined to be 8.2 s-1. Since the plasmid is described by a radius of gyration of 0.22 microns, the intradomain concentration of base pairs could be calculated. Using this concentration (180 microM), the rate constant for intradomain nonspecific association of RNA polymerase to pAR1319 DNA was estimated to be 4.6 x 10(4) M-1 s-1. In addition, a mathematical model has been used to fit the other two important rate constants to the experimental data: koff, which describes the dissociation of RNA polymerase from nonspecific binding sites, and D1, the one-dimensional diffusion coefficient of the enzyme along the DNA molecule. In this model, the circular DNA molecule is described as a ring of interconnected binding sites which together comprise a DNA "domain." RNA polymerase, which enters the domain via three-dimensional diffusion and binds to each site, is allowed to diffuse linearly between adjacent sites and three-dimensionally on and off the DNA molecule. The rate equations for the time-dependent occupancy of each site by RNA polymerase could be written, based on general principles. By solving the resulting family of differential equations, koff and D1 were determined to be 0.3 s-1 and 1.5 x 10(-9) cm2 s-1, respectively.     

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.     

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.     

Multi-step fibrinogen binding to the integrin (alpha)IIb(beta)3 detected using force spectroscopy

The regulated ability of integrin alphaIIbbeta3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. We have developed a model system based on laser tweezers, enabling us to measure specific rupture forces needed to separate single receptor-ligand complexes. First of all, we performed a thorough and statistically representative analysis of nonspecific protein-protein binding versus specific alphaIIbbeta3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified alphaIIbbeta3 and fibrinogen, covalently attached to underlying surfaces, ranged from approximately 20 to 150 pN. This distribution could be fit with a sum of an exponential curve for weak to moderate (20-60 pN) forces, and a Gaussian curve for strong (>60 pN) rupture forces that peaked at 80-90 pN. The interactions corresponding to these rupture force regimes differed in their susceptibility to alphaIIbbeta3 antagonists or Mn2+, an alphaIIbbeta3 activator. Varying the surface density of fibrinogen changed the total binding probability linearly >3.5-fold but did not affect the shape of the rupture force distribution, indicating that the measurements represent single-molecule binding. The yield strength of alphaIIbbeta3-fibrinogen interactions was independent of the loading rate (160-16,000 pN/s), whereas their binding probability markedly correlated with the duration of contact. The aggregate of data provides evidence for complex multi-step binding/unbinding pathways of alphaIIbbeta3 and fibrinogen revealed at the single-molecule level.     

BstYI bound to noncognate DNA reveals a "hemispecific" complex: implications for DNA scanning

DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA.