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.     

Herpes simplex virus type 1 single-strand DNA binding protein ICP8 enhances the nuclease activity of the UL12 alkaline nuclease by increasing its processivity

UL12 is a 5'- to 3'-exonuclease encoded by herpes simplex virus type 1 (HSV-1) which degrades single- and double-stranded DNA. UL12 and the single-strand DNA binding protein ICP8 mediate a strand exchange reaction. We found that ICP8 inhibited UL12 digestion of single-stranded DNA but stimulated digestion of double-stranded DNA threefold. The stimulatory effect of ICP8 was independent of a strand exchange reaction; furthermore, the effect was specific to ICP8, as it could not be reproduced by Escherichia coli single-stranded DNA binding protein. The effect of ICP8 on the rate of UL12 double-stranded DNA digestion is attributable to an increase in processivity in the presence of ICP8.     

Quantifying DNA-protein interactions by double-stranded DNA arrays.

We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.     

A simple method for the preparation of single-stranded polydeoxynucleotides of desired length.

A method for the preparation of homogeneous, single-stranded polydeoxynucleotides of desired length up to 800 bases is described. The procedure entails 1) generation of double-stranded DNA of desired length by PCR using a pair of primers of which one is biotinylated and the other is either unlabeled or fluorescently labeled, 2) isolation of PCR products by agarose slab gel electrophoresis, 3) recovery of desired product by electroelution, 4) binding of the product to streptavidin-coated magnetic beads and is followed by 5) duplex denaturation and removal of the unbound single strand that is either unlabeled or fluorescently labeled. Final product characteristics were determined by capillary gel electrophoresis with fluorescence detection. Up to microgram quantities of homogeneous single-stranded DNA of a desired length were obtained. These can be used as single-stranded size standards in capillary gel electrophoresis experiments as well as in other techniques requiring such standards.     

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.     

Progress towards single-molecule DNA sequencing: a one color demonstration

Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.     

Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.     

Structure of reaction intermediates formed during Saccharomyces cerevisiae Rad51-catalyzed strand transfer

The process by which the Saccharomyces cerevisiae strand transfer protein, Rad51, seeks out homologous sequences in vivo can be modeled by an in vitro reaction between a single-stranded DNA circle and a double-stranded linear DNA. In addition to the substrates and products, electrophoresis of reaction mixtures resolves two groups of low mobility bands. Here we show that the low mobility bands formed during strand transfer by Rad51 (or Escherichia coli RecA) represent joint molecules (JM) between the two substrates. One group, which we name JM1, is an obligatory reaction intermediate in which the complementary strand from the duplex substrate has been partially transferred to the single-stranded circle. Our assignment is based on pulse-chase and restriction enzyme digestion experiments and verified by electron microscopy. The slower moving group of bands, designated JM2, is formed by an unexpected reaction between JM1 and a second double-stranded linear substrate. Strand transfer of the second duplex initiates noncanonically from the end where the complementary strand is recessed. Thus JM2 is formed by two strand transfer reactions with the same single-stranded circular substrate but with opposite polarities. Finally, we show that the multiple sharp bands in JM1 and JM2 are the result of substrate sequences that pause strand transfer.     

Nucleosomal DNA is digested to repeats of 10 bases by exonuclease III

Nucleosomes were treated with increasing concentrations of exonuclease III (Exo III) from E. coli. At low levels of Exo III, the heterogeneous distribution of monomers (with associated DNA fragments ranging in size between 140 and 170 bp) is "trimmed" down to a discrete core of 140 bp. The "trimming" of monomers to 140 bp results from a 3' exonucleolytic digestion accompanied by a 5' clipping activity which is specific for the conformation of internucleosomal DNA. At higher concentrations of Exo III, the enzyme digests the 140 bp "trimmed" nucleosome core from both 3' ends without associated 5' nuclease activity. Most striking is the observation that the fragments produced during such a digestion display discrete single-stranded lengths that are integer multiples of 10 bases. For some dimer nucleosomes, Exo III can digest as many as 200 bases from at least one 3' end and produce a 10 base interval ladder from about 400 bases down to 180 bases. This suggests that the enzyme can traverse the length of an entire nucleosome without destroying whatever structural features are necessary to produce a 10 base DNA ladder.     

Direct observation of strand passage by DNA-topoisomerase and its limited processivity

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51 ± 0.33 μm (11 ± 6 turns) of a braid was unlinked in a burst of reactions taking 8 ± 4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25-37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ~100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ~10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ~10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.     

Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage λ gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.     

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.     

A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase

Mitochondrial DNA polymerase (pol gamma) is the sole DNA polymerase responsible for replication and repair of animal mitochondrial DNA. Here, we address the molecular mechanism by which the human holoenzyme achieves high processivity in nucleotide polymerization. We have determined the crystal structure of human pol gamma-beta, the accessory subunit that binds with high affinity to the catalytic core, pol gamma-alpha, to stimulate its activity and enhance holoenzyme processivity. We find that human pol gamma-beta shares a high level of structural similarity to class IIa aminoacyl tRNA synthetases, and forms a dimer in the crystal. A human pol gamma/DNA complex model was developed using the structures of the pol gamma-beta dimer and the bacteriophage T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol gamma-beta and the human catalytic core that allow it to encircle the newly synthesized double-stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Biochemical properties of a novel set of human pol gamma-beta mutants are explained by and test the model, and elucidate the role of the accessory subunit as a novel type of processivity factor in stimulating pol gamma activity and in enhancing processivity.     

Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III

Escherichia coli topoisomerases I and III can decatenate double-stranded DNA (dsDNA) molecules containing single-stranded DNA regions or nicks as well as relax negatively supercoiled DNA. Although the proteins share a mechanism of action and have similar structures, they participate in different cellular processes. Whereas topoisomerase III is a more efficient decatenase than topoisomerase I, the opposite is true for DNA relaxation. In order to investigate the differences in the mechanism of these two prototypical type IA topoisomerases, we studied DNA decatenation at the single-molecule level using braids of intact dsDNA and nicked dsDNA with bulges. We found that neither protein decatenates an intact DNA braid. In contrast, both enzymes exhibited robust decatenation activity on DNA braids with a bulge. The experiments reveal that a main difference between the unbraiding mechanisms of these topoisomerases lies in the pauses between decatenation cycles. Shorter pauses for topoisomerase III result in a higher decatenation rate. In addition, topoisomerase III shows a strong dependence on the crossover angle of the DNA strands. These real-time observations reveal the kinetic characteristics of the decatenation mechanism and help explain the differences between their activities.     

Visualizing the assembly of human Rad51 filaments on double-stranded DNA

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into extended nucleoprotein filaments on DNA. To study the dynamic behavior of Rad51 we have developed a single-molecule assay that relies on a combination of hydrodynamic force and microscale diffusion barriers to align individual DNA molecules on the surface of a microfluidic sample chamber that is coated with a lipid bilayer. When visualized with total internal reflection fluorescence microscopy (TIRFM), these "molecular curtains" allow for the direct visualization of hundreds of individual DNA molecules. Using this approach, we have analyzed the binding of human Rad51 to single molecules of double-stranded DNA under a variety of different reaction conditions by monitoring the extension of the fluorescently labeled DNA, which coincides with assembly of the nucleoprotein filament. We have also generated several mutants in conserved regions of Rad51 implicated in DNA binding, and tested them for their ability to assemble into extended filaments. We show that proteins with mutations within the DNA-binding surface located on the N-terminal domain still retain the ability to form extended nucleoprotein filaments. Mutations in the L1 loop, which projects towards the central axis of the filament, completely abolish assembly of extended filaments. In contrast, most mutations within or near the L2 DNA-binding loop, which is also located near the central axis of the filament, do not affect the ability of the protein to assemble into extended filaments on double-stranded (ds)DNA. Taken together, these results demonstrate that the L1-loop plays a crucial role in the assembly of extended nucleoprotein filaments on dsDNA, but the N-terminal domain and the L2 DNA-binding loop have significantly less impact on this process. The results presented here also provide an important initial framework for beginning to study the biochemical behaviors of Rad51 nucleoprotein filaments using our novel experimental system.     

Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement

A single copy of bacteriophage T7 DNA polymerase and DNA helicase advance the replication fork with a processivity greater than 17,000 nucleotides. Nonetheless, the polymerase transiently dissociates from the DNA without leaving the replisome. Ensemble and single-molecule techniques demonstrate that this dynamic processivity is made possible by two modes of DNA polymerase-helicase interaction. During DNA synthesis the polymerase and the helicase interact at a high-affinity site. In this polymerizing mode, the polymerase dissociates from the DNA approximately every 5000 bases. The polymerase, however, remains bound to the helicase via an electrostatic binding mode that involves the acidic C-terminal tail of the helicase and a basic region in the polymerase to which the processivity factor also binds. The polymerase transfers via the electrostatic interaction around the hexameric helicase in search of the primer-template.     

Correlated cleavage of single- and double-stranded substrates by uracil-DNA glycosylase

Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double- and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.     

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine.

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.