New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis.

We describe improve enzymatic methods for sequencing method for sequencing DNA. They are based on partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3' ends shortened to varying lengths, followed by repair synthesis to extend and label the 3' ends. After asymmetrical cleavage of the DNA with a restriction enzyme, the labeled products are separated by gel electrophoresis and the sequence read from the autoradiogram. The entire procedures, beginning with unrestricted DNA and followed through gel electrophoresis, takes only one day for sequencing both strands of the DNA molecule. These methods are especially suitable for sequencing DNA cloned in plasmid vectors, and they greatly extend the usefulness of the dideoxynucleotide chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463, 1977). Using these methods we have determined the sequence of a 410 base pair fragment which includes the yeast SUP3 tyrosine tRNA gene.     

Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.     

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.     

Locking the DNA gate of DNA gyrase: investigating the effects on DNA cleavage and ATP hydrolysis

Supercoiling by DNA gyrase involves the passage of one segment of double-stranded DNA through another. This requires a DNA duplex to be cleaved and the broken ends separated by at least 20 A. This is accomplished by the opening of a dimer interface, termed the DNA gate, which is covalently attached to the broken ends of the DNA. After strand passage, the DNA gate closes allowing the reunion of the broken ends. We have cross-linked the DNA gate of gyrase using cysteine cross-linking to block gate opening. We show that this locked gate mutant can bind quinolone drugs and perform DNA cleavage. However, locking the DNA gate prevents strand passage and the ability of DNA to stimulate ATP hydrolysis. We discuss the mechanistic implications of these results.     

Use of a chemically modified T7 DNA polymerase for manual and automated sequencing of supercoiled DNA

Procedures are presented for reliable and accurate nucleotide sequence analysis using as template supercoiled DNA prepared by a modified rapid boiling minipreparation protocol. This method yields DNA templates suitable for sequencing within 1 h of bacterial harvest. We describe optimal reaction conditions for supercoiled miniprep DNA sequencing using a modified T7 DNA polymerase (Sequenase) in dideoxynucleotide chain termination reactions. We demonstrate that under these conditions, the sequencing data obtained with miniprep DNA is indistinguishable from that obtained with CsCl purified supercoiled DNA or from that obtained using single stranded DNA templates. We further show that the supercoiled DNA sequencing reactions can be analyzed on a commercially available automated DNA sequencing system that detects 32P labeled DNA during its electrophoretic separation. Taken together, these developments represent a significant improvement in the process of nucleotide sequence analysis.     

Vectors containing infrequently cleaved restriction sites for use in BAL 31 nuclease-assisted and end-label-mediated analysis of cloned DNA fragments

Cloning vectors derived from plasmids pUC8 and pUC18 and phage M13mp10 were constructed so as to have multiple cloning sites (MCS) flanked by the recognition/cleavage sites for the Sfi I and Not I restriction nucleases. Cleavage of vectors containing cloned DNA fragments with either of the infrequently cleaving Sfi I or Not I endonucleases will usually yield linear DNAs cleaved only at the corresponding site in the MCS, so that the cloned insert can be degraded unidirectionally by the duplex exonuclease activity of the BAL 31 nucleases until an amount equal to the length of the vector has been degraded. The ends of the above constructs resulting from cleavage with Not I or Sfi I can readily be labeled, with labeling at only the terminus of the cloned DNA available for the Sfi I site. The BAL 31 nuclease-mediated procedures enhance a previous technique for mapping of restriction enzyme fragments, allow for localization of sequences in cloned segments for which a probe is available, and improve a method for sequencing cloned inserts through the production of sets of nested unidirectional deletions from either end of the parent cloned fragment. The advantages of end-label-mediated restriction site mapping using the above vectors over existing such procedures are also demonstrated.     

Preferential modification of guanine bases in DNA by dimethyldioxirane and its application to DNA sequencing

From gel sequencing experiments with 32P-end-labelled oligodeoxyribonucleotides, it is shown that treatment of DNA with the powerful oxidant dimethyldioxirane, followed by heating in piperidine, causes selective strand scission at the sites of guanine bases. The same specificity for cleavage at guanine was observed with a 45-mer labelled at either the 3'- or 5'-end and with a single and double stranded 34-mer. On account of its speed and operational simplicity, modification with dimethyldioxirane is proposed as a practicable alternative to conventional chemical sequencing procedures for locating guanine bases in DNA.     

Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu

Transposition of Mu involves transfer of the 3' ends of Mu DNA to the 5' ends of a staggered cut in the target DNA. We find that cleavage at the 3' ends of Mu DNA precedes cutting of the target DNA. The resulting nicked species exists as a noncovalent nucleoprotein complex in which the two Mu ends are held together. This cleaved donor complex completes strand transfer when a target DNA, Mu B protein, and ATP are provided. Mu end DNA sequences that have been precisely cut at their 3' ends by a restriction endonuclease, instead of by Mu A protein and HU, are efficiently transferred to a target DNA upon subsequent incubation with Mu A protein, Mu B protein, and ATP. Cleavage of the Mu ends therefore cannot be energetically coupled with joining these ends to a target DNA. We discuss the DNA strand transfer mechanism in view of these results, and propose a model involving direct transfer of the 5' ends of the cut target DNA, from their original partners, to the 3' ends of Mu.     

Structure of bleomycin-induced DNA double-strand breaks: predominance of blunt ends and single-base 5' extensions

In order to examine the structure of bleomycin-induced DNA double-strand breaks, defined-sequence DNA was labeled in each strand at a single restriction site and treated with bleomycin. Various double-stranded fragments resulting from bleomycin-induced double-strand breaks were isolated, denatured, and run on sequencing gels to determine the sites of cleavage in each strand. For virtually every double-strand break, the cleavage site in one strand was a pyrimidine in a G-Py sequence, reflecting a specificity similar to that of bleomycin-induced single-strand cleavage. However, the cleavage site in the complementary strand was seldom a G-Py sequence, and was usually a site where single-strand cleavage was infrequent. When the sequence at the double-strand break was G-Py-Py', the break at Py was usually accompanied by a break at the base directly opposite Py, resulting in blunt ends. When the sequence was G-Py-Pu, the break at Py was usually accompanied by a break at the base opposite Pu, resulting in single-base 5' extensions. Double-strand breaks with 3' extensions, such as would result from cleavage of two C residues in a self-complementary G-C sequence, were conspicuously absent. These data provide further evidence that bleomycin-induced double-strand breaks do not result from coincidence of independent site-specific single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)     

A photoinduced cleavage of DNA useful for determining T residues.

Irradiation of 5'-[32P]-phosphate labeled DNA fragments with ultraviolet light in the presence of primary amines followed by piperidine treatment resulted in base-specific cleavage of the DNA chain at T residues, accompanied by a less intensive G reaction. This simple, T greater than G cleavage offers an alternative method for determining T residues in chemical DNA sequencing.     

Rapid mapping of restriction sites of a DNA: restriction of agarose gel and two-dimensional analysis of end-labeled DNA.

A new two-dimensional technique for the mapping of restriction sites is presented. Linear DNA labeled at both ends is first partially digested with the restriction endonuclease for which a map is desired. Following electrophoresis of the partial digest in an agarose gel, complete digestion of the fragments in the gel matrix with a second restriction enzyme is carried out. Electrophoresis in the second dimension resolves two sets of labeled spots: one set from the left and the other from the right end. For a given band of the autoradiogram of the first dimension gel, the mobility of the band gives the size of the DNA fragment, and therefore the distance of a particular restriction site from one of the ends of the original linear DNA. The mobility of the labeled spot derived from this band in the second dimension gel allows one to distinguish whether the distance deduced above is from one end or the other. Additional information about the location of one set of restriction sites for one enzyme relative to those for a second enzyme can also be obtained using the two-dimensional method. The advantages of the technique are the small amount of DNA required and the rapidity with which many maps can be constructed from one labeled DNA. As a test of the method, maps for the HindIII and HaeIII cleavage sites of circular phage PM2 DNA have been obtained, after first converting the DNA to the linear form by digestion with HpaII.     

Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension

Identification of nucleotides used for RNA chain initiation or for contacting DNA binding proteins is basic to our understanding of gene regulation. Normally, a radioactive primer is used to copy RNA or DNA. The polymerase extension stops at free ends of mRNA (as in promoter mapping) or at the position of template cleavage or modification (as in footprinting). The locations of these positions are then analyzed by polyacrylamide gel electrophoresis. These analyses have been improved using fluorescently labeled primers and commonly available DNA sequencing machines. The protocol, which we call fluorescently labeled oligonucleotide extension (FLOE), eliminates the need for handling radioactivity and polyacrylamide. The DNA sequencer delivers data as a "trace" that is ready for quantification, which eliminates the need to trace gels separately. The data analysis is further improved by new software, Scanalyze, which we present here. We demonstrate that by using promoter mapping and footprinting, FLOE shortens experimental time, extends the stretch of analyzable sequence, and simplifies quantification compared to radioactive methods and is as sensitive in terms of detecting templates.     

Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects

We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.     

HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.

Retroviral DNA integration involves a coordinated set of DNA cutting and joining reactions. Linear viral DNA is cleaved at each 3' end to generate the precursor ends for integration. The resulting recessed 3' ends are inserted into target DNA by a subsequent DNA strand transfer reaction. Purified HIV-1 integration protein carries out both of these steps in vitro. Two novel forms of the dinucleotide cleaved from HIV-1 DNA were identified and one, a cyclic dinucleotide, was used to analyze the stereochemical course of viral DNA cleavage. Both viral DNA cleavage and DNA strand transfer display inversion at chiral phosphorothioates during the course of the reaction. These results suggest that both reactions occur by a one-step mechanism without involvement of a covalent protein-DNA intermediate.     

On the DNA cleavage mechanism of Type I restriction enzymes

Although the DNA cleavage mechanism of Type I restriction–modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5′- and 3′-overhangs of varying lengths. EcoAI preferentially generated 3′-overhangs of 2–3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5′-overhangs of a length of ~6–7 and 3–5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.     

DNA damage induced by ascorbate in the presence of Cu2+

DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates. Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min. The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end. DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+. Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage. The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random. These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment. These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.     

Purification of mitochondrial and plastid DNA

The size, structure and conformation of mitochondrial and plastid genomes differ dramatically among eukaryotes. Similarly, the yield and purity of extracted organelle DNA also vary, and are crucial factors for the success of restriction mapping and sequencing experiments. We describe here procedures for the purification of organelle DNA from a broad range of eukaryotes. By emphasizing the underlying principles, these procedures will facilitate the development of new species-specific protocols. The presented purification schemes involve either isolation of organelles and subsequent extraction of DNA from this subcellular fraction, or processing of whole-cell lysates followed by CsCl gradient centrifugation to separate nuclear and organelle DNAs according to their A + T content. We have successfully used the described procedures for organelle genome sequencing from diverse eukaryotes, including non-axenic protists. Procedures can be completed in 3-5 days, typically yielding a few micrograms of DNA-ample for sequencing complete genomes.     

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.     

Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation

Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation. The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data.     

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.