Preparation of DNA catenanes and observation of their topological structures by atomic force microscopy.

DNA catenanes have been prepared by the reaction of T4 DNA ligase with linear DNA in the presence of nicked DNA. Single molecular images of DNA catenanes and large circular DNAs have been clearly observed by AFM using a tapping mode at room temperature and in an ambient atmosphere.     

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.     

The old exonuclease of bacteriophage P2.

The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.     

Method to eliminate linear DNA from mixture containing nicked circular, supercoiled, and linear plasmid DNA

Preparations of circular plasmid DNA in either supercoiled or nicked circular form often are contaminated with undesired linear DNA fragments arising from shearing/degradation of chromosomal DNA or linearization of plasmid DNA itself. We report a simple enzymatic method, using a combination of λ exonuclease and RecJ_f, for the selective removal of linear DNA from such mixtures. λ exonuclease digests one strand of linear duplex DNA in the 5′ to 3′ direction, whereas RecJ_f, a single-strand-specific exonuclease, digests the remaining complementary single strand into mononucleotides. This combination of exonucleases can remove linear DNA from a mixture of linear and supercoiled DNA, leaving the supercoiled form intact. Furthermore, the inability of λ exonuclease to initiate digestion at nicks or gaps enables the removal of undesired linear DNA when nicked circular DNA has been enzymatically prepared from supercoiled DNA. This method can be useful in the preparation of homogeneous circular plasmid DNA required for therapeutic applications and biophysical studies.     

Simian virus 40 minichromosomes contain torsionally strained DNA molecules.

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.     

An improved procedure for measuring DNA replication with transient assays in eukaryotic cells

We describe an improved method for quantifying covalently closed circular DNAs replicated in eukaryotic cells. Normally, for such assays, plasmid DNAs transfected into animal cells must be extensively digested with restriction enzymes such as Dpnl and the products of digestion of the input DNA separated from the Dpnl-resistant (replicated) DNAs by electrophoresis. In the procedure described, the input, unreplicated DNA is nicked by light digestion with Dpnl and subsequently denatured and eliminated by S1 nuclease digestion. The replicated Dpnl-resistant DNA is detected by slot blot hybridization. Multiple samples can be rapidly assayed, with a considerable reduction in expense and labor.     

Analysis of polyoma virus DNA replicative intermediates by agarose gel electrophoresis.

Agarose gel electrophoresis has been used to fractionate polyoma virus DNA replicative intermediates (RI) according to maturity. Approximate electrophoretic mobility versus maturity relationships were obtained for both intact (supercoiled) and nicked (relaxed) RI. There was considerable overlap between the supercoiled and relaxed RI populations after electrophoretic fractionation. Intact RI could be recovered from preparative agarose gels for further analysis by centrifugation, electron microscopy, re-electrophoresis, or nuclease digestion.     

Role of the DNA ligase III zinc finger in polynucleotide binding and ligation.

Mammalian DNA ligase III exists as two distinct isoforms denoted alpha and beta. Both forms possess a motif that is homologous to the putative zinc finger present in poly(ADP-ribose) polymerase. Here, the role of this motif in the binding and ligation of nicked DNA and RNA substrates in vitro has been examined in both isoforms. Disruption of the putative zinc finger did not affect DNA ligase III activity on nicked DNA duplex, nor did it abolish DNA ligase III-alpha activity during DNA base excision repair in a cell-free assay. In contrast, disruption of this motif reduced 3-fold the activity of both DNA ligase III isoforms on nicked RNA present in RNA/DNA homopolymers. Furthermore, whereas disruption of the motif did not prevent binding of DNA ligase III to nicked DNA duplex, binding to nicked RNA homopolymers was reduced approximately 10-fold. These results suggest that the putative zinc finger does not stimulate DNA ligase III activity on simple nicked DNA substrates, but indicate that this motif can target the binding and activity of DNA ligase III to nicked RNA homopolymer. The implications of these results to the cellular role of the putative zinc finger are discussed.     

The measurement of exonuclease activities by atomic force microscopy

We have applied atomic force microscopy (AFM) to the measurement of BAL 31 nuclease activities. BAL 31 nuclease, a species of exonuclease, is used to remove unwanted sequences from the termini of DNA before cloning. For cutting out only the appropriate sequences, it is important to know the nuclease properties, such as digestion speed and the distribution of the lengths of the digested DNA. AFM was used to obtain accurate measurements on the lengths of DNA fragments before and after BAL 31 nuclease digestion. We analyzed 4 DNAs with known number of base pairs (288, 778, 1818, and 3162 base pairs) for correlating the contour length measured by AFM with the number of base pairs under the deposition conditions used. We used this calibration for analyzing DNA degradation by BAL 31 nuclease from the AFM measurement of contour lengths of digested DNAs. In addition, the distribution of digested DNA could be analyzed in more detail by AFM than by electrophoresis, because digested DNA were measured as a population by electrophoresis, but were measured individually by AFM. These results show that AFM will be a useful new technique for measuring nuclease activities. Received: 8 August 1997 / Accepted: 10 September 1997     

Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro.

Trichosanthin, an abortifacient, immunosuppressive and anti-tumor protein purified from the traditional Chinese herb medicine Tian Hua Fen, is a potent inhibitor against HIV-1 replication. Under normal enzymatic digestion conditions, trichosanthin cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA. Trichosanthin has no effect on linear double-stranded DNA. Neither does it convert relaxed circular duplex DNA into a supercoiled form in the presence of ATP. Thus trichosanthin is not a DNA gyrase. However, trichosanthin can cleave the relaxed circular DNA into a linear form, indicating that both the circular as well as the supercoiled forms are essential for trichosanthin recognition. In addition, trichosanthin contains one calcium metal ion per protein molecule, which presumably is related to its endonucleolytic activity.     

Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection

In chimpanzee hepatitis B virus (HBV) carriers, the mechanism of viral persistence has been examined by analyzing viral DNA molecules in liver and serum. Chimpanzee liver DNA contained two extrachromosomal HBV DNA molecules migrating on hybridization blots at 4.0 kb and 2.3 kb. There was no evidence for integration of HBV DNA into the host genome. The extrachromosomal molecules were distinct from Dane particle DNA and were converted to linear 3.25 kb full-length double-stranded HBV DNA on digestion with Eco RI. Nucleases S1 and Bal 31 converted "2.3 kb" HBV DNA to 3.25 kb via an intermediate of "4.0 kb" apparent length. The HBV DNA molecule that migrated at 2.3 kb represents a supercoiled form I of the HBV genome, and the molecule that migrated at 4.0 kb represents a full-length "nicked," relaxed circular form II. Evidence for supercoiled HBV DNA in serum Dane particles was obtained by production of form II molecules upon digestion with nuclease S1 or Bal 31. It is proposed that most Dane particles represent interfering noninfectious virus containing partially double-stranded DNA circles and that particles containing supercoiled HBV DNA may represent infectious hepatitis B virus.     

Hydrolysis by restriction endonucleases at their DNA recognition sequences substituted with mismatched base pairs.

Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA. These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA. None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions. However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site. The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site. In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs.     

Direct observation of the assembly of RecA/DNA complexes by atomic force microscopy

The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATPgammaS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 x 10(-5) RecA min(-1) bp(-1). The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min(-1). These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.     

Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I.

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.     

Bacteriophage T4 endonuclease II: concerted single-strand nicks yield double-strand cleavage

In vivo, endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated. In vitro, however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks. Upper strand nick positions vary both in vivo and in vitro. A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII. Using DNA ligase in vitro, we found that EndoII nicked both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site. This indicates that the right-side CSE at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage. Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand. In addition, the upper and lower strands were nicked independently of each other, seldom resulting in double-strand cleavage. Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.     

Characterization of viral genomes in the liver and serum of chimpanzee long-term hepatitis B virus carriers: a possible role for supercoiled HBV-DNA in persistent HBV infection

In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV-DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV-DNA molecule which migrated at 4.0 kb position represents a full-length "nicked," relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV-DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV-DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence are discussed.     

Deoxyribonucleolytic activity of alpha- and beta-momorcharins.

alpha- and beta-Momorcharins were purified by an improved procedure using the affinity Affi-gel Blue gel and the ion exchange Mono-S FPLC column. Both purified alpha- and beta-momorcharins possessed deoxyribonucleolytic activity. Under normal digestion conditions, they cleaved the supercoiled, double-stranded SV-40 DNA to produce nicked circular and linear DNAs. Prolonged incubation did not have any further effects. On the other hand, the linear DNAs, lambda, Ad-2 and T7 were not digested by alpha- nor beta-momorcharin. Thus, it appears that the conformation of the DNA may be the determining factor for the deoxyribonucleolytic activity of these momorcharins.     

Electron microscopic studies of bacteriophage M13 DNA replication.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (theta, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in theta molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of theta to other RF molecules does not change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.     

Survival of M13mp18 gapped duplex DNA as a function of gap length

Gaps of various lengths were generated in duplex M13mp18DNA by exonuclease III digestion of nicked DNA. The length of the gap increased essentially linearly with time of digestion. Survival in E. coli, however, was not a linear function of gap length. Similar results were obtained when gaps were produced by stopping the polymerization reaction. The survival (N/No) of the gapped DNA in SOS-induced E. coli cells transformed by electroporation and uninduced cells transformed by the calcium chloride method can be quantitatively accounted for by a kinetic model assuming a single-strand endonucleolytic activity (Pd) in the cell which increases linearly with gap length (L) and a repair activity by a polymerase (Pr) which is independent of gap length (formula 1). With uninduced cells transformed by electroporation the results can be mathematically described if assumptions are made concerning the protection of single-stranded parts of the DNA by single-strand affinic proteins.