A method for isolation of a large amount of a single-stranded DNA fragment

Single-stranded DNA inserts can be digested from recombinant phage DNA of M13mp7 with BamHI or EcoRI restriction endonucleases. The single-stranded DNA is satisfactory for DNA sequencing and nuclei acid hybridization.     

Uptake of homologous single-stranded fragments by superhelical DNA: II. Characterization of the reaction

We have studied the association of superhelical DNA (RFI)³ of phage G4 with defined single-stranded fragments isolated after cleavage of viral (+) strands by endonuclease R · HaeIII. The sedimentation rates of complexes formed by uptake of different single-stranded restriction fragments by G4 RFI were consistent with the view that base-pairing between the two components causes unwinding of superhelical turns, with one negative superhelical turn removed for every ten nucleotide residues of third strand taken up. The combining ratio of superhelical DNA and a single specific fragment was close to unity.At high concentrations of salt, nitrocellulose filters efficiently retained complexes of superhelical DNA and homologous fragments, which provided the basis for a rapid assay, and permitted the estimation of the thermodynamic and kinetic parameters of strand uptake in vitro. The reaction is reversible, with an apparent K_eq of approximately 10⁶m^?1. Apparent rate constants, k₁, for uptake of different fragments (85 to 1100 nucleotides long) varied about fourfold, with no obvious relationship to the length of the fragment. In 10 mm-Tris · HCl (pH 7.5), 200 mm-NaCl, fragments were taken up most rapidly at about 75 °C. Under these conditions, the apparent k₁ for a fragment 250 nucleotides long was approximately 600 m^?1s^?1, which is two or three orders of magnitude slower than the calculated rate of association of complementary strands of that length. At physiological temperatures, appreciable rates of strand uptake were seen only at low concentrations of salt (4 mm-Na⁺ in 10 mm-Tris · HCl), and were one or two orders of magnitude less than the rate at 75 °C in 200 mm-NaCl. At a given concentration of counterion a threshold temperature exists above which the rate of reaction rises sharply from an undetectable level.Thermodynamic calculations indicate that the reaction is entropically driven, and that the rate is limited by a step exhibiting a positive entropy and enthalpy of activation. The data are consistent with a model for strand uptake in which the rate-limiting step is the unstacking of a small number of base-pairs in the superhelical DNA. Stabilization and extension of the nucleus of base-pairs formed with the incoming strand is favored by the decrease in free energy associated with removal of superhelical turns.     

Interruptions in Single Strands of the DNA in Slime Mold and Other Organisms

The molecular weight of single-stranded DNA from the slime mold Physarum polycephalum has been determined by alkaline gradient centrifugation. The average molecular weight during DNA synthesis (~1.5 × 10⁷ D) is less than that observed in nonsynthetic periods (~4 × 10⁷ D). On the basis of a chromosome number of 50 per nucleus and a DNA content of 1 μμg per nucleus, we are led to conclude that at pH 12 each chromosome dissociates into 300 (single-stranded) pieces of DNA. We have also compared the sedimentation profiles of single-stranded DNA from Escherichia coli, PPLO, and T2 bacteriophage. These data support the conjecture that each bacterial chromosome can be dissociated into 10 or 12 single-stranded pieces of DNA. Dissociation of DNA into multiple pieces under our experimental conditions is best interpreted in terms of interruptions in the continuity of the DNA either by naturally occurring gaps or at alkali-labile bonds.     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

A circular channel crucible oscillating viscometer: Detection of DNA damage induced in vivo by exceedingly small doses of dimethylnitrosamine

A new oscillating crucible viscometer, having a U-shaped circular channel, is described. The damping coefficient δ is lowered by an increase of the viscosity η. The instrument described here allows the solution to come in contact with inert plastic only. At all steps of its preparation and during viscosity measurements, giant DNA from rat liver nuclei was maintained at shear stresses around 10^?4 dynes cm^?2. Viscosity was studied as a function of surface tension, DNA concentration and shear stress. It was found that under our experimental conditions it was possible to obtain meaningful values for reduced viscosity, η_red, practically identical to intrinsic viscosity [η]. Rat liver nuclei are incubated in an alkaline lysing solution (pH 12.5; 22 °C): they are lysed immediately and the released DNA starts to uncoil. The viscosity of solutions of this giant DNA increases very slowly with time, reaching a maximum only after about ten hours. The process was accelerated by single-stranded breaks arising from methylation of DNA in vivo with dimethylnitrosamine. It was found that the time of DNA disentanglement was sensitive to an exceedingly small number of breaks. We think that we were able to measure molecular weights around the length of the single strand of an average chromosome (M_n 5 × 10¹⁰). An empirical relation between molecular weight and reduced viscosity after complete disentanglement was also established, as a linear log-log plot, covering a molecular weight range between 10⁸ and 2.5 × 10¹⁰. It is suggested that the viscosimetric evaluation of DNA disentanglement is probably the most sensitive method for studying DNA damage induced “in vivo” by chemical carcinogens.     

DNA synthetic activity of nuclei isolated from differentiating cardiac muscle and association of DNA polymerase alpha with the outer nuclear membrane

[³H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [³H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [³H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication.     

Post-replication DNA repair in barley embryos treated with N-methyl-N-nitrosourea

In sterile cultures of free barley embryos, N-methyl-N-nitrosourea (MNU) caused a decrease in the size of both template [¹⁴C]-labeled DNA and of daughter [³H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [³H]thymidine incorporation. In addition, duplexes containing [³H]-daughter DNA analyzed in BND cellulose contained more single-stranded regions in MNU-treated embryos than in the corresponding control. Incubation of MNU-treated embryos in nutrient medium for up to 18 h after the [³H]-labeling permitted the recovery of small-sized daughter DNA to full-sized strands and led to the enhancement of double-strandedness of DNA duplexes containing [³H]-labeled strands. If [³H]-labeling had been carried out 8–10 h after the MNU treatment, the size of daughter DNA, the proportion of double-strandedness and the rate of thymidine uptake into DNA partially increased in comparison with rates observed when labeling had been done just after or 3 h after the MNU treatment, but these variables did not reach the values of the corresponding controls.     

Effect of nuclear Ca2+ uptake inhibitors on Ca2+-activated DNA fragmentation in rat liver nuclei

The effect of nuclear Ca²⁺ uptake inhibitors on the Ca²⁺-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca²⁺ (40 M) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca²⁺-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca²⁺ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 M arachidonic acid, 2.0 mM NAD⁺, 10 M zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca²⁺-ATPase activity and Ca²⁺ uptake in the nuclei, produced a significant inhibition of the Ca²⁺-activated DNA fragmentation in the nuclei. The results show that the Ca²⁺-activated DNA fragmentation is involved in the uptake of Ca²⁺ by the nuclei, suggesting a role of Ca²⁺ transport system in the regulation of liver nuclear functions.     

RPA is an initiation factor for human chromosomal DNA replication

The initiation of chromosomal DNA replication in human cell nuclei is not well understood because of its complexity. To allow investigation of this process on a molecular level, we have recently established a cell-free system that initiates chromosomal DNA replication in an origin-specific manner under cell cycle control in isolated human cell nuclei. We have now used fractionation and reconstitution experiments to functionally identify cellular factors present in a human cell extract that trigger initiation of chromosomal DNA replication in this system. Initial fractionation of a cytosolic extract indicates the presence of at least two independent and non-redundant initiation factors. We have purified one of these factors to homogeneity and identified it as the single-stranded DNA binding protein RPA. The prokaryotic single-stranded DNA binding protein SSB cannot substitute for RPA in the initiation of human chromosomal DNA replication. Antibodies specific for human RPA inhibit the initiation step of human chromosomal DNA replication in vitro. RPA is recruited to DNA replication foci and becomes phosphorylated concomitant with the initiation step in vitro. These data establish a direct functional role for RPA as an essential factor for the initiation of human chromosomal DNA replication.     

Formation of Single-Stranded DNA during DNA Transformation of Neisseria gonorrhoeae

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.     

DdrB stimulates single-stranded DNA annealing and facilitates RecA-independent DNA repair in Deinococcus radiodurans

The bacterium Deinococcus radiodurans can survive extremely high exposure to ionizing radiation. The repair mechanisms involved in this extraordinary ability are still being investigated. ddrB is one gene that is highly up-regulated after irradiation, and it has been proposed to be involved in RecA-independent repair in D. radiodurans. Here we cloned, expressed and characterized ddrB in order to define its roles in the radioresistance of D. radiodurans. DdrB preferentially binds to single-stranded DNA. Moreover, it interacts directly with single-stranded binding protein of D. radiodurans DrSSB, and stimulates single-stranded DNA annealing even in the presence of DrSSB. The post-irradiation DNA repair kinetics of a ddrB/recA double mutant were compared to ddrB and recA single mutants by pulsed-field gel electrophoresis (PFGE). DNA fragment rejoining in the ddrB/recA double mutant is severely compromised, suggesting that DdrB-mediated single-stranded annealing plays a critical role in the RecA-independent DNA repair of D. radiodurans.     

Structure and function of TatD exonuclease in DNA repair

TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life. It has been suggested that TatD participates in DNA fragmentation during apoptosis in eukaryotic cells. However, the cellular functions and biochemical properties of TatD in bacterial and non-apoptotic eukaryotic cells remain elusive. Here we show that Escherichia coli TatD is a Mg²⁺-dependent 3′–5′ exonuclease that prefers to digest single-stranded DNA and RNA. TatD-knockout cells are less resistant to the DNA damaging agent hydrogen peroxide, and TatD can remove damaged deaminated nucleotides from a DNA chain, suggesting that it may play a role in the H₂O₂-induced DNA repair. The crystal structure of the apo-form TatD and TatD bound to a single-stranded three-nucleotide DNA was determined by X-ray diffraction methods at a resolution of 2.0 and 2.9 Å, respectively. TatD has a TIM-barrel fold and the single-stranded DNA is bound at the loop region on the top of the barrel. Mutational studies further identify important conserved metal ion-binding and catalytic residues in the TatD active site for DNA hydrolysis. We thus conclude that TatD is a new class of TIM-barrel 3′–5′ exonuclease that not only degrades chromosomal DNA during apoptosis but also processes single-stranded DNA during DNA repair.     

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho^–] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho^–] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.     

Programmable cleavage of linear double-stranded DNA by combined action of Argonaute CbAgo from Clostridium butyricum and nuclease deficient RecBC helicase from E. coli

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Currently, the use of mesophilic pAgos as programmable endonucleases is hampered by their limited action on double-stranded DNA (dsDNA). We demonstrate here that efficient cleavage of linear dsDNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated in vitro via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecB^exo–C). Properties of CbAgo and characteristics of simultaneous cleavage of DNA strands in concurrence with DNA strand unwinding by RecB^exo–C were thoroughly explored using 0.03–25 kb dsDNAs. When combined with RecB^exo–C, CbAgo could cleave targets located 11–12.5 kb from the ends of linear dsDNA at 37°C. Our study demonstrates that CbAgo with RecB^exo–C can be programmed to generate DNA fragments with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. The combination of CbAgo and RecB^exo–C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for in vitro use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of linear dsDNA at any desired location.     

A freeze-squeeze method for recovering long DNA from agarose gels

Long DNA can be recovered from agarose gels after electrophoresis by freezing the gel slices and manually squeezing out liquid containing the DNA. With this method the recoveries of phage T₇ DNA (molecular weight 25 × 10⁶) and the open and closed forms of circular phage PM2 DNA (molecular weight 6 × 10⁶) were about 70%. Sedimentation analysis shows that the extruded DNA has not sustained double- or single-stranded breaks. The extruded DNA can be used without further purification as substrate for the restriction endonuclease Hind_II,III, from Hemophilus influenzae, for DNA·DNA hybridization and for electron microscopy.     

Interaction of poly-L-lysine with chromatin. Inhibition of in situ priming for Escherichia coli DNA polymerase

The priming capacity of chromatin of fixed nuclei and chromosomes for exogenous DNA polymerase can be evaluated radioautographically by the incorporation of labeled nucleotides. It had previously been reported that acid fixation or acid treatment of alcohol-fixed tissues led to increased priming when calf thymus DNA polymerases, specific for single-stranded DNA, were used. We employed Escherichia coli DNA polymerase and sequential treatments of the fixed tissue with acid and poly-L-lysine in order to elucidate the mechanism through which the acid effect is produced. Acid treatment enhanced chromatin priming for the E coli DNA polymerase, and saturation of the chromatin with poly-L-lysine strongly inhibited the reaction. This inhibition was reversible through subsequent treatment with acid. Wide differences in priming were observed between cell types of alcohol-fixed chicken blood smears: thrombocyte and lymphocyte nuclei exhibited strong priming ability whereas erythrocyte nuclei failed to support any detectable priming. We conclude that the acid effect is readily interpretable in terms of acid-mediated changes in the association between DNA and protein in the chromatin complex.     

Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA

Methylglyoxal, a known endogenous and environmental mutagen, is a reactive α-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a ‘primed template’ DNA and the exonuclease-deficient Klenow fragment (KF^exo–) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS–PAGE, we found that methylglyoxal induced a DNA–KF^exo– crosslink. The specific binding complex of KF^exo– and ‘primed template’ DNA was necessary for formation of the DNA–KF^exo– crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2′-deoxyguanosine was incubated with Nα-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA–DNA polymerase crosslink.     

Replication of bacteriophage M13. XII. In vivo cross-linking of a phage-specific DNA binding protein to the single-stranded DNA of bacteriophage M13 by ultraviolet irradiation.

Ultraviolet irradiation of bacteriophage M13-infected Escherichia coli induces the formation of a covalent crosslink between progeny single-stranded DNA and the M13 DNA binding protein, the product of gene 5. The crosslinked complex is readily isolated from detergent-treated lysates by sucrose-gradient velocity sedimentation and CsCl equilibrium sedimentation in the presence of detergent. The crosslinked complex produced with optimal levels of irradiation sediments 1.06 times faster than uncomplexed M13 single-stranded DNA, has a buoyant density of approximately 1.62 to 1.64 g/cm³ and a protein to DNA mass ratio of 2 mg protein per mg DNA. Cleavage of the crosslinked complex with cyanogen bromide or trypsin yields products similar to those produced by cleavage of purified M13 gene 5 protein. The crosslink is located close to the carboxyl terminus of the protein.     

Restricted uptake of ethidium bromide and propidium diiodide by denatured closed circular DNA in buoyant cesium chloride

Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.     

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3′-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.