Real-time observation of a single DNA digestion by lambda exonuclease under a fluorescence microscope field

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-protein interactions, such as the DNA digestion reaction by lambda exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual lambda exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lambda exonuclease, a stained lambda DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by lambda exonuclease revealed that the DNA digestion rate was approximately 1000 bases/s and also demonstrated high processivity.     

Dynamic molecular combing: a 'low tech' method for genetic mapping and diagnosis

So much of molecular genetics involves invisible samples at the bottom of minuscule tubes, that the attraction of something you can actually see is virtually irresistible. Recent developments in high-resolution fluorescent in situ hybridization (FISH) are both visually appealing and a valuable addition to the repertoire of gene-mapping and diagnostic techniques. The latest of these, dubbed dynamic molecular combing allows precise, high-resolution mapping of markers on cloned or total genomic DNA. In this technique, a coverslip coated with silane is dipped into a DNA solution. After a few minutes' incubation, during which the ends of the DNA molecules bind to the coverslip, a small motorized device is used to withdraw the coverslip vertically, by its edge, at a constant rate of 300 microseconds⁻¹. The force exerted on the DNA molecules by the meniscus stretches all the molecules uniformly in the same direction, and they dry instantly onto the silanized surface as it is exposed to air. The result is a surface covered with a high density of parallel DNA molecules, which can then be probed with fluorescent-labelled markers and visualized under a miscroscope. Because of the constant stretching factor of 2 kb per micrometer of fibre length, no internal control is needed for calibration, unlike other recent fibre-FISH methods. In pilot experiments, the technique has been used successfully to measure microdeletions involving the tuberous sclerosis 2 gene, for fine-mapping and orienting of sets of contiguous cosmid clones (contigs) on a yeast artificial chromosome within its yeast genome, and to measure gaps in a cosmid contig by hybridizing pairs of cosmid probes to combed total human genomic DNA. Dynamic molecular combing will also be useful for a variety of applications in genetic diagnosis, such as mapping the breakpoints where segments of different chromosomes have been interchanged, and analysing sequence rearrangements within genes. Probes as small as 3 kb can be used when mapping cloned DNA; when total genomic DNA is the template, however, the probe needs to be at least 5 kb to enable the signal to be distinguished from background. The length of DNA that can be visualized in any one field of view, approximately 400-600 kb, is determined by the characteristics of the camera used to record the image and the objective on the microscope. The new equipment needed is simple and the protocol very straightforward, which should put it within the reach of most genetics laboratories. The motorized device used to withdraw the coverslip from the DNA solution at the required speed of 300 micrometer s⁻¹ will be available commercially from early 1998 (contact the authors of the Science paper for details). Alternatively, such a device could be readily constructed by most departmental workshops. The silane-coated coverslips are best prepared in the gaseous phase, especially for combing total genomic DNA, when a homogeneous surface is essential. The researchers in Paris and London who developed the technique are, therefore, also considering making the coverslips commercially available.     

A new method for straightening DNA molecules for optical restriction mapping.

We have developed an improved method of straightening DNA molecules for use in optical restriction mapping. The DNA was straightened on 3-aminopropyltriethoxysilane-coated glass slides using surface tension generated by a moving meniscus. In our method the meniscus motion was controlled mechanically, which provides advantages of speed and uniformity of the straightened molecules. Variation in the affinity of the silanized surfaces for DNA was compensated by precoating the slide with single-stranded non-target blocking DNA. A small amount of MgCl2 added to the DNA suspension increased the DNA-surface affinity and was necessary for efficient restriction enzyme digestion of the straightened surface-bound DNA. By adjusting the amounts of blocking DNA and MgCl2, we prepared slides that contained many straight parallel DNA molecules. Straightened lambda phage DNA (48 kb) bound to a slide surface was digested by EcoRI restriction endonuclease, and the resulting restriction fragments were imaged by fluorescence microscopy using a CCD camera. The observed fragment lengths showed excellent agreement with their predicted lengths.     

Study of microorganism genome DNA by atomic force microscopy

The potential of atomic force microscopy (AFM) for the investigation of peculiarities of microorganisms genome structure is demonstrated. AFM images of phage lambda DNA linear molecules and supercoiled mica in buffer solution was imaged in air. New experimental method of DNA stretching based on using amino-modified mica with a decreased surface density of active amino-groups is proposed. Stretched molecules of phage lambda DNA were imaged by AFM.     

PNA microarrays for hybridisation of unlabelled DNA samples

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.     

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).Open in a separate windowClick here to view.^{(103M, flv)}     

Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide-thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl-oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.     

Method for orienting DNA molecules on mica surfaces in one direction for atomic force microscopy imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.     

Capsid transformation during packaging of bacteriophage lambdaDNA.

Assembly pathways of complex viruses might not be simple additions of one protein after another with rigid tertiary structure. It might in fact involve shifts in subunit structure, movement of subunits relative to each other to form new arrangements, transient action of proteins and protein segments, involvement of structure forming 'microenvironments' of the host. Thus morphogenesis of the bacteriophage lambda head starts with the formation of a core-containing DNA-free petit lambda particle. In a first transition, and dependent on a host function, the core is released, minor protein components of the capsid are processed and the particle's structure is altered, as shown by a change of its hydrodynamic properties. The resulting 'prehead' undergoes a second transition triggered by a complex of DNA and recognition protein (A-protein). This transition is more drastic than the first one. The particle doubles its volume without increasing in protein mass, the shell becomes thinner, and the surface structure is changed. Concomitantly with this process, the DNA becomes packaged and the particle becomes able to bind the small 'D-protein' in amounts equimolar to the capsid protein, which it could not do before. The D-protein addition probably causes another shift of the capsid structure. DNA packaging is completed, and the DNA is cut from concatemeric precursors to unit length molecules. Binding sites are created for the tail connector molecules which in turn allow the independently assembled tail to attach. Research on these processes proceeds along several lines: comparison of physical and chemical properties of particles accumulating in mutants; pulse-chase experiments on assembly precursors; morphogenesis in vitro; and model transitions of aberrant lambda polyheads.     

Packing of binding stained DNA strands in bacteriophage lambda

Distribution of stainable DNA strands in phage lambda has been studied by polarized fluorescence. The effect of tight DNA-packing on fluorescence depolarization of complex dye-DNA was calculated. It is shown that stainable DNA in the phage is not concentrated in the central region. The arrangement of acridine orange molecules on the surface layers of the packed DNA is the most probable one.     

A simple method for freeze-drying of macromolecules and macromolecular complexes

We present a simple approach for effective freeze-drying and rotary shadowing of large molecules, molecular assemblies, and cell organelles. Simply, a suspension of specimen is adsorped to a glass coverslip, stabilized, and rinsed with 30% methanol. A second coverslip is "sandwiched" on top, and excess methanol is withdrawn from the edges then frozen by plunging into liquid nitrogen and split. Following either rotary or unidirectional shadowing and replication, the coverslip is dissolved in hydrofluoric acid. In addition to avoiding the problems encountered with air-drying specimens for rotary shadowing, the technique also reproducibly provides the thin layer of solution necessary for proper freeze-drying, regardless of how hydrophobic the sample is. The "glass sandwich" technique allows modification of the glass substrate (making it hydrophobic with carbon or hydrophilic by soaking it in alcian blue) which clearly alters the shape of macromolecular assemblies such as myosin filaments and decorated thin filaments.     

Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins.

Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed 'grpE-dependent'. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda-lambda O structure. The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.     

Method for stretching DNA molecules on mica surface in one direction for AFM imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. The immobilization process was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were shown to be fully stretched in one direction on imaging them by AFM.     

Visualization of a specific sequence on a single large DNA molecule using fluorescence microscopy based on a new DNA-stretching method.

A method for analyzing large DNA which makes it possible to obtain spatial information on the positions of specific sequences along a DNA molecule has been developed. Making use of the fact that large DNA molecules are stably elongated under an alternating-current field in a concentrated linear polymer solution, the direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. The spatial positions of the fluorescent spots of the probe (fluorescence-labeled restriction endonuclease EcoRI) on DNA molecules were determined by image analysis. As expected, fluorescent spots of EcoRI were observed at certain positions on lambda DNA, where sequences to which EcoRI binds are located. Finally, the potential application of single large DNA molecule analysis using this DNA-stretching method is discussed.     

Template-directed interference footprinting of cytosine contacts in a protein-DNA complex: potent interference by 5-aza-2'-deoxycytidine.

In the template-directed interference (TDI) footprinting method (Hayashibara & Verdine, 1990), analogs of the naturally occurring DNA bases are incorporated into DNA enzymatically and assayed for interference of sequence-specific binding by a protein. Here we extend this method to include analysis of contacts of amino acid residues to the major groove surface of cytosine residues (TDI-C footprinting). The base analog 5-aza-2'-deoxycytidine, in which the hydrophobic 5-CH of cytosine is replaced by a hydrophilic aza nitrogen, was incorporated into DNA via the corresponding 5'-triphosphate. The analog was found to base pair with guanine during polymerization, resulting in substitution of 2'-deoxycytidine residues. TDI-C footprints of the lambda repressor-OL1 operator complex revealed apparent contacts to the cytosines at operator positions 7 and 8. Inspection of the high-resolution X-ray crystal structure of the lambda-OL1 complex (Clarke et al., 1992; Beamer & Pabo, 1992) revealed that C8 makes a hydrogen binding contact with the Lys3; C7, on the other hand, makes a previously unnoticed hydrophobic contact with the alkane side chain of Lys3. In only the consensus operator half-site was cytosine interference observed, suggesting that the nonconsensus arm binds DNA very differently if at all. The N-terminal arm represents the archetypal case of a sequence-specific peptide-DNA complex characterized at high resolution; thus, the present studies suggest strategies for design and screening of DNA binding peptides. The finding that 5-aza-2'-deoxycytidine inhibits sequence-specific DNA binding proteins may suggest an alternative rationale for the biological activities of this and related azapyrimidine nucleosides.     

Combing DNA on CTAB-coated surfaces

A fluorescence microscope (FM) coupled with an intensified charge-coupled device (ICCD) camera was used to investigate the combing of DNA on cetyltrimethyl ammonium bromide (CTAB)-coated glass surfaces. DNA molecules can be combed uniform and straight on CTAB-coated surfaces. Different combing characteristics at different pH values were found. At lower pH (ca. 5.5), DNA molecules were stretched 30% longer than the unextended and DNA extremities bound with CTAB-coated surfaces via hydrophobic interaction. At high pH values (e.g., 6.4 and 6.5), DNA molecules were extended about 10% longer and DNA extremities bound with CTAB-coated surfaces via electrostatic attraction. At pH 6.0, DNA molecules could be extended 30% longer on 0.2-mM CTAB-coated surfaces. CTAB cationic surfactant has both a hydrophobic motif and a positively charged group. So, CTAB-coated surfaces can bind DNA extremities via hydrophobic effect or electrostatic attraction at different pH values. It was also found that combing of DNA on CTAB-coated surfaces is reversible. The number of DNA base pairs binding to CTAB-coated surfaces was calculated.     

Molecular cloning of covalently closed circular DNA of bovine leukemia virus

The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs.     

Lambda bacteriophage-mediated transduction of ColE1 deoxyribonucleic acid having a lambda bacteriophage-cohesive end site: selection of packageable-length deoxyribonucleic acid.

An in vitro recombinant ColE1-cos lambda deoxyribonucleic acid (DNA) molecule, pKY96, has 70% of the length of lambda phage DNA. The process of lambda phage-mediated transduction of pKY96 generated a small amount of transducing phage particles containing ColE1-cos lambda DNA molecules of 80 or 101% of the length of lambda phage DNA, in addition to those containing original pKY96 DNA molecules. The newly isolated larger plasmid DNAs were transduced 100 times more efficiently than pKY96 DNA. Their structures were compared with that of a prototype pKY96 DNA, and the mechanism of the formation of these molecules is discussed.