Direct probing: covalent attachment of probe DNA to double-stranded target DNA.

We report a novel procedure, which can be applied to probing of specific DNA, for covalently attaching probe DNA to complementary sequences in double-stranded target DNA. Employing hairpin-like oligonucleotide probes in combination with successive use of recA protein and DNA ligase, probes can be attached directly to target DNA molecules without dissociation of the DNA. The hairpin-like structure of the probes was designed so that the terminus of the probe oligonucleotide can be brought into close stereochemical proximity to the terminus of the complementary strand of target DNA for ligation. Because of the elimination of the DNA dissociation and subsequent hybridization (and washing) steps in the currently employed method, the probing process has become greatly simplified and more efficient and may lead to development of fully automated probing systems.     

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3'-ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.     

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.     

Determination of the relative positions of amino acids by partial specific cleavages of end-labeled proteins

We have developed a new method for obtaining information about protein sequences that uses an approach analogous to that used to determine DNA sequences. In essence, three steps are involved. First, a detectable label is attached exclusively to the amino terminus of a polypeptide. Next, the labeled chain is subjected to partial specific cleavage in a way that produces roughly equimolar amounts of fragments of different sizes. Cleavages for methionine, tryptophan, arginine, aspartyl-proline bonds, and asparaginyl-glycine bonds have been employed. Lastly, the labeled fragments are separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of target amino acids along the polypeptide chain can be deduced from the specific pattern of labeled bands by reading the "ladder" in the same way that DNA sequencing gels are read. Although the method can be conducted with a radioactive label, we have chosen to use a fluorescent label. We have applied the method successfully to the three subunit chains of two different fibrinogens.     

End-labeling of long DNA fragments with biotin and detection of DNA immobilized on magnetic beads

To immobilize DNA fragments onto magnetic beads coated with streptavidin for isolation purpose, it is important to label one biotin molecule at one terminus of DNA fragment. After failure to label long DNA with biotin by PCR and filling-in reaction, a 9.2 kb DNA was labeled with biotin by a modified ligation strategy. A simple method is also reported to detect the quantity and integrity of DNA immobilized on the magnetic beads.     

Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli

The RecBC enzyme of Escherichia coli promotes genetic recombination of phage or bacterial chromosomes. The purified enzyme travels through duplex DNA, unwinding and rewinding the DNA with the transient production of potentially recombinogenic single-stranded DNA. The studies reported here are aimed at understanding which chromosomal forms allow the entry of RecBC enzyme and hence may undergo RecBC enzyme-mediated recombination. Circular duplex molecules, whether covalently closed, nicked or containing single-stranded gaps of 10 to 774 nucleotides, are not detectably unwound by RecBC enzyme. Linear duplex molecules are readily unwound if they have a nearly flush-ended terminus whose 5' and 3' ends are offset by no more than about 25 nucleotides; molecules with longer single-stranded tails are poorly bound by RecBC enzyme and are infrequently unwound. The single-strand endonuclease activity of RecBC enzyme can slowly cleave gapped circles to produce molecules presumably capable of being unwound. These results provide an enzymatic basis for the recombinogenicity of double-stranded DNA ends established from genetic studies of RecBC enzyme and Chi sites, recognition sites for RecBC enzyme-mediated DNA strand cleavage.     

Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase II. Potential roles in recombination.

Drosophila melanogaster topoisomerase II is capable of joining phi X174 (+) strand DNA that it has cleaved to duplex oligonucleotide acceptor molecules by an intermolecular ligation reaction (Gale, K. C. and Osheroff, N. (1990) Biochemistry 29, 9538-9545). In order to investigate potential mechanisms for topoisomerase II-mediated DNA recombination, this intrinsic enzyme activity was further characterized. Intermolecular DNA ligation proceeded in a time-dependent fashion and was concentration-dependent with respect to oligonucleotide. The covalent linkage between phi X174 (+) strand DNA and acceptor molecules was confirmed by Southern analysis and alkaline gel electrophoresis. Topoisomerase II-mediated intermolecular DNA ligation required the oligonucleotide to contain a 3'-OH terminus. Moreover, the reaction was dependent on the presence of a divalent cation, was inhibited by salt, and was not affected by the presence of ATP. The enzyme was capable of ligating phi X174 (+) strand DNA to double-stranded oligonucleotides that contained 5'-overhang, 3'-overhand, or blunt ends. Single-stranded, nicked, or gapped oligonucleotides also could be used as acceptor molecules. These results demonstrate that the type II enzyme has an intrinsic ability to mediate illegitimate DNA recombination in vitro and suggests possible roles for topoisomerase II in nucleic acid recombination in vivo.     

Multiple topological labeling for imaging single plasmids

Sequence-specific labeling methods for double-stranded DNA are required for mapping protein binding sites or specific DNA structures on circular DNA molecules by high-resolution imaging techniques such as electron and atomic force microscopies. Site-specific labeling can be achieved by ligating a DNA fragment to a stem-loop-triplex-forming oligonucleotide, thereby forming a topologically linked complex. The superhelicity of the plasmid is not altered and the process can be applied to two different target sites simultaneously, using DNA fragments of different sizes. Observation of the labeled plasmids by electron microscopy revealed that, under conditions where the triple helices were stable, the two labels were located at 339+/-34 bp from one another, in agreement with the distance between the two target sequences for triple helix formation (350 bp). Under conditions where the triple helices were not stable, the short DNA fragments could slide away from their target site. The concomitant attachment of two different stable labels makes it possible, for the first time to our knowledge, to label a circular DNA molecule and obtain information on its direction. In addition to its potential applications as a tool for structural investigations of single DNA molecules and their interactions with proteins, this DNA labeling method may also prove useful in biotechnology and gene therapy.     

Methylated DNA labels for marking objects

We recently described a method for digitally labelling objects with DNA. Here we show that, using DNA methyltransferases to create polymorphic DNA templates, it is possible to significantly increase the number of labels that can be generated by this method. Nine double-stranded DNA templates of different length were methylated with either M.HaeIII or M.AluI methyltransferase, or both. Different mixtures of methylated and unmethylated versions of this template set were used to 'invisibly' label paper. The mixtures were eluted from the paper and the methylated status of the templates in each mixture successfully determined, and the labels read, by digestion with the complementary restriction endonuclease, followed by a polymerase chain reaction and agarose gel electrophoresis. One methylated DNA label was read after it had been left on paper for two months.     

Sequence independent duplex DNA opening reaction catalysed by SV40 large tumor antigen.

Simian virus 40 (SV40) large tumor antigen (T antigen) is mainly localized in the nucleus where it exhibits two biochemical properties: DNA binding and helicase activity. Both activities are necessary for viral DNA replication and may also enable T antigen to modulate cellular growth. Here we present biochemical and electron microscopic evidence that the helicase activity can start at internal sites of fully double-stranded DNA molecules not containing the SV40 origin or replication. Using T antigen specific monoclonal antibodies, this unwinding reaction can be biochemically divided in an initiation (duplex opening) and a propagation step. The duplex opening reaction (as well as the propagation step) does not depend on a specific DNA sequence or secondary structure. In addition, we have found that T antigen forms an ATP dependent nucleoprotein complex at double-stranded DNA, which may be an essential step for the sequence independent duplex DNA opening reaction.     

Replication of polyoma DNA in isolated nuclei: analysis of replication fork movement.

The movement of replication forks during polyoma DNA synthesis in isolated nuclei was analyzed by digesting newly synthesized DNA with the restriction endonuclease HpaII which cleaves polyoma DNA into eight unique fragments. The terminus of in vitro DNA synthesis was identified by cleaving newly completed molecules with HpaII. The distribution of label in the restriction fragments showed that the in vitro DNA synthesis was bidirectional and had the normal terminus of replication. Analysis of replicative intermediates pulse-labeled in vitro further suggested that DNA synthesis in isolated nuclei is an ordered process similar to replication in intact cells. Replication forks moved with a constant rate from the origin towards the terminus of replication. The nonlinear course of the DNA synthesis reaction in the isolated nuclei seems to result from the random inactivation of replication forks rather than a decrease in the rate of fork movement. During the in vitro synthesis a replication fork could maximally synthesize a DNA chain about 1,000 nucleotides long. The results suggest that some replication forks might be initiated in vitro at the origin of replication.     

3'-5' exonuclease of Klenow fragment: role of amino acid residues within the single-stranded DNA binding region in exonucleolysis and duplex DNA melting

The mechanism of the 3'-5' exonuclease activity of the Klenow fragment of DNA polymerase I has been investigated with a combination of biochemical and spectroscopic techniques. Site-directed mutagenesis was used to make alanine substitutions of side chains that interact with the DNA substrate on the 5' side of the scissile phosphodiester bond. Kinetic parameters for 3'-5' exonuclease cleavage of single- and double-stranded DNA substrates were determined for each mutant protein in order to probe the role of the selected side chains in the exonuclease reaction. The results indicate that side chains that interact with the penultimate nucleotide (Q419, N420, and Y423) are important for anchoring the DNA substrate at the active site or ensuring proper geometry of the scissile phosphate. In contrast, side chains that interact with the third nucleotide from the DNA terminus (K422 and R455) do not participate directly in exonuclease cleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, and R455 have markedly different effects on the cleavage of single- and double-stranded DNA, causing a much greater loss of activity in the case of a duplex substrate. Time-resolved fluorescence anisotropy decay measurements with a dansyl-labeled primer/template indicate that the Q419A, Y423A, and R455A mutations disrupted the ability of the Klenow fragment to melt duplex DNA and bind the frayed terminus at the exonuclease site. In contrast, the N420A mutation stabilized binding of a duplex terminus to the exonuclease site, suggesting that the N420 side chain facilitates the 3'-5' exonuclease reaction by introducing strain into the bound DNA substrate. Together, these results demonstrate that protein side chains that interact with the second or third nucleotides from the terminus can participate in both the chemical step of the exonuclease reaction, by anchoring the substrate in the active site or by ensuring proper geometry of the scissile phosphate, and in the prechemical steps of double-stranded DNA hydrolysis, by facilitating duplex melting.     

Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs.

A cell-free system from Xenopus eggs mimics the reaction occurring at the eukaryotic replicative fork in vivo when chromatin assembly is coupled to complementary strand synthesis of DNA. DNA synthesis on single-stranded circular DNA promotes supercoiling and the replicated molecule sediments as a discrete nucleoprotein complex. Micrococcal nuclease digestion reveals a characteristic pattern of multiples of 200 bp of DNA. The kinetics of chromatin assembly and DNA synthesis are coincident and both processes occur with a rate comparable with chromosomal replication in vivo in early embryos. The DNA synthesis reaction can be uncoupled from the assembly reaction. Thus, titration of chromatin proteins by preincubation of the extract with double-stranded DNA prevents the supercoiling of replicated DNA without affecting the rate of synthesis. In contrast, chromatin assembly performed on unreplicated double-stranded DNA is a slower process as compared with the assembly coupled to DNA synthesis. Supercoiled molecules are detected after 30 min replication whereas at least 2 h are required to observe the first form I DNA with unreplicated double-stranded DNA. Such a system where chromatin assembly is promoted by DNA synthesis should be valuable for studying the interaction of specific factors with DNA during chromatin assembly at the replicative fork.     

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem–loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 ± 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.     

Strand-invading linear probe combined with unmodified PNA

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40 °C at 100 mM NaCl concentration.     

Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.     

Nucleotide sequence of the self-priming 3'' terminus of the single-stranded DNA extracted from the parvovirus Kilham rat virus.

The parvovirus genome is a linear, single-stranded DNA molecule with double-stranded hairpin termini. The 3' terminus can serve in vitro as a self-primer for the synthesis of a double-stranded viral DNA intermediate. We have sequenced the nucleotides in the 3' terminus and propose a model for the secondary structure of the terminus and the in vitro origin of replication for the complementary viral DNA strand.