Improvement of efficiency of the Ce(IV)-induced DNA scission--relationship between the kinetic parameters (k(cat) and Km) and the DNA structure.

The Michaelis constant (Km) for double-stranded DNA, single-stranded DNA, and dinucleotide hydrolysis by Ce(IV) ion are 4.4, 15, and more than 40 mM, respectively. The order of the k(cat), however, is dinucleotide > oligonucleotides. Not only the improvement of k(cat) but also that of Km is important for the design of an efficient artificial nuclease.     

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson–Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37°C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.     

Preferential hydrolysis of gap and bulge sites in DNA by Ce(IV)/EDTA complex

A new strategy for site-selective DNA hydrolysis, which takes advantage of the difference in reactivity between the phosphodiester linkages at the target site and the others, is presented. As the molecular scissors, homogeneous Ce(IV)/ethylenediamine-N,N,N′,N′-tetraacetate (EDTA) complex is used without being bound to any sequence-recognizing moiety. When a gap structure is formed at the target site by using two short oligonucleotides and the composite is treated with the Ce(IV)/EDTA complex at pH 7.0 and 37°C, the gap site in the substrate DNA is preferentially hydrolyzed over the double-stranded portion of the DNA. Site-selective DNA scission is also achieved by forming a bulge structure at the target site with the use of the appropriate oligonucleotide. These site-selective scissions are based on the following two factors: (i) the phosphodiester linkages in a single-stranded DNA are far more susceptible to the hydrolysis by the Ce(IV) complex than are the linkages in double-stranded DNA, and (ii) the phosphodiester linkages in the bulge sites are still more reactive than those in single-stranded DNA. In both cases, the addition of spermine significantly accelerates the scission.     

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.     

Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage.

Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN). IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro. Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity. The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA. A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited. The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN. Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA. Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA. IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.     

Structural requirements of double and single stranded DNA substrates and inhibitors, including a photoaffinity label, of Fpg protein from Escherichia coli

Fpg protein (formamidopyrimidine or 8-oxoguanine DNA glycosylase) from E. coli catalyzes excision of several damaged purine bases, including 8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine from DNA. In this study the interaction of E. coli Fpg with various specific and nonspecific oligodeoxynucleotides was analyzed. Fpg was shown to remove 8-oxoguanine efficiently, not only from double-stranded, but also from single-stranded oligodeoxynucleotides. The Michaelis constants (KM) of a range of single-stranded oligodeoxynucleotides (0.55-1.3 microM) were shown to be 12-170 times higher that those for corresponding double-stranded oligodeoxynucleotides (KM = 6-60 nM). Depending on the position of the 8-oxoguanine within the oligodeoxynucleotides, relative initial rates of conversion of single-stranded substrates were found to be lower than, comparable to, or higher than those for double-stranded oligodeoxynucleotides. The enzyme can interact effectively not only with specific, but also with nonspecific single-stranded and double-stranded oligodeoxynucleotides, which are competitive inhibitors of the enzyme towards substrate. Fpg became irreversibly labeled after UV-irradiation in the presence of photoreactive analogs of single-stranded and double-stranded oligodeoxynucleotides. Specific and nonspecific single-stranded and double-stranded oligodeoxynucleotides essentially completely prevented the covalent binding of Fpg by the photoreactive analog. All these data argue for similar interactions occurring in the DNA binding cleft of the enzyme with both specific and nonspecific oligodeoxynucleotides. The relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides differ by no more than 2 orders of magnitude. Addition of the second complementary chain increases the affinity of the first single-stranded chain by a factor of approximately 10. It is concluded that Michaelis complex formation of Fpg with DNA containing 8-oxoG cannot alone provide the major part of the enzyme specificity, which is found to lie in the kcat term for catalysis; the reaction rate being increased by 6-7 orders of magnitude by the transition from nonspecific to specific oligodeoxynucleotides.     

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.     

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA breaks and nicks and the consequent enzyme activation. Other structures recognize intact DNA

The recognition of double-stranded DNA breaks and single-stranded nicks by human poly(ADP-ribose) polymerase and the consequent enzymic activation were examined using derivatives of the enzyme expressed in Escherichia coli. The N-terminal 162 residues encompass two zinc fingers. Deletion or mutation of the first finger results in a loss of activation by DNA with either single-stranded or double-stranded damage. Destruction of the second finger reduces activation by double-stranded DNA breaks only slightly, but eliminates activation by single-stranded DNA nicks. These data suggest that activation by single-stranded DNA nicks requires two zinc fingers, but activation by double-stranded DNA breaks requires only the finger closer to the N terminus. Variant proteins that lack both zinc fingers are enzymically inactive but still exhibit weak DNA binding, which is independent of DNA damage. Thus, other regions are also capable of binding intact DNA, but the recognition of a strand nick or break which occasions the synthesis of poly(ADP-ribose) specifically requires the zinc fingers.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

A Streptomyces glaucescens endodeoxyribonuclease which shows a strong preference for CC dinucleotide.

We have characterized a deoxyribonuclease from Streptomyces glaucescens that cleaves double-stranded DNA preferably between the dinucleotide 5'-CC-3'. The cleavage specificity was demonstrated by both analysis of the terminal nucleotides of the generated fragments and DNA sequencing of partially digested DNA. Digestion of lambda DNA with this enzyme resulted in the production of double-stranded fragments with 5' and/or 3'-protruding single-stranded tails. DNase I footprinting experiments indicated that the nuclease specifically binds to its cleavage sites on the DNA under non-catalytic conditions. The enzyme is not affected by cytosine methylation in hemimethylated DNA.     

Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA.

A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA. This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing. First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR). Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer is approximately 0.4 microM. Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions. The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing. This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA.     

Affinity and Conformation of the Estrogen Receptor When Bound to Single-Stranded vs. Double-Stranded Nonspecific DNA

Abstract

The affinity of the hormone-bound estrogen receptor for single-stranded and double-stranded DNA was compared using isocratic elution chromatography. The receptor bound single-stranded DNA with a two-fold higher affinity than double-stranded DNA (17.9 × 10⁴ M^?1 vs. 9.1 × 10⁴ M^?1) at 0.2 M KCl. The same number of ions were released when the receptor bound either single-stranded or double-stranded DNA (11.8 vs. 10.6, respectively). These results indicate the hormone-bound estrogen receptor has no strong preference for single-stranded vs. double-stranded nonspecific DNA, and has a similar conformation when bound to either form of DNA at physiological salt concentrations.     

Topoisomerase action on short DNA duplexes reveals requirements for gate and transfer DNA segments

Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50-60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.     

Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated exonuclease activities.

Purified DNA polymerase III has two distinct exonuclease activities: one initiates hydrolsis at the 3 termini, and the other at the 5 termini of single-stranded DNA. Both exonucleases have the same relative mobility on polyacrylamide gels as the polymerase activity. Molecular identity of the three activities is further indicated by their comparative rates of thermal inactivation and their sensitivity to ionic strength. The 3-5 exonuclease activity hydrolyzes only single-standed DNA. The rate of hydrolysis is twice the optimal rate of polymerization. The products are 5-mononucleotides, but the 3-5 activity is unable to cleave free dinucleotides or the 5-terminal dinucleotide of a polydeoxynucleotide chain. The 3-5 activity will not degrade 3-phosphoryl-terminated oligonucleotides such as d(pTpTpTp). The 5-3 activity catalyzes the hydrolysis of single-stranded DNA at 1/15 the rate of the 3-5 exonuclease. The 5-3 exonuclease requires the presence of a 5 single-stranded terminus in order to initiate hydrolysis, but will thereafter proceed into a double-stranded region. Although the limit products found during hydrolysis of substrates designed to assay specifically the 5-3 activity are predominantly mono- and dinucleotides, these products probably arise from the subsequent hydrolysis of oligonucleotides by the 3-5 hydrolytic activity. This interpretation is supported by (a) the relatively greater activity of the 3-5 exonuclease, (b) the inability of the enzyme to degrade d(pTpTpTp), and (c) the release of the 5 terminus of a single-stranded DNA molecule as an oligonucleotide. The 5-3 exonuclease attacks ultraviolet-irradiated duplex DNA which has first been incised by the Micrococcus luteus endonuclease specific for thymine dimers in DNA.     

Recognition of single-stranded DNA by the bacteriophage T4-induced type II topoisomerase

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.     

Forms of Deoxyribonucleic Acid Produced by Virions of the Ribonucleic Acid Tumor Viruses

The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs(2)SO(4) gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.     

Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage

Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the −1 position, i.e. immediately 5′ of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria.