Transfection of REP- mycoplasmas with viral single-stranded DNA.

Double-stranded DNA from mycoplasma virus L2 can transfect Acholeplasma laidlawii cells in the presence of polyethylene glycol (T. L. Sladek and J. Maniloff, J. Bacteriol. 155:734-741, 1983). We report here that both single-stranded DNA and double-stranded replicative form DNA, from the single-stranded DNA mycoplasma virus L51, are also infectious in this system. For both DNAs transfection frequencies were in the range of 10(-8) transfectants per DNA molecule and 10(-3) transfectants per CFU. An unexpected finding was that both DNAs could transfect A. laidlawii strain REP-, a variant which is a nonpermissive host for single-stranded DNA mycoplasma viruses due to a block in viral DNA replication (Nowak et al., J. Bacteriol. 127:832-836, 1976). The number of viruses produced by transfected REP- cells was comparable to the number produced by both transfected and infected wild-type cells. Therefore, transfected L51 DNAs are able to bypass the replication block in REP- cells that occurs when these cells are infected by L51 virions.     

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.     

Intracellular localization of rat-liver uracil-DNA glycosylase. Purification and properties of the chromatin enzyme

Most of the uracil-DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil-DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibited by increasing ionic strength and free uracil. The apparent Km is independent of the strandedness of the DNA substrate containing uracil, but V is slightly higher with the single-stranded substrate. The frequency of uracil substitution in the double-stranded DNA influences the kinetic parameters: a higher frequency increases both Km and V. The inhibitory effects of NaCl and free uracil are greater when the substrate is double-stranded rather than single-stranded. It is speculated that, acting either on the DNA or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme-substrate complex. The increased reaction rate with a higher frequency of uracil residues in double-stranded DNA is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that processivity also occurs with single-stranded DNA and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single-stranded substrate has a higher uracil content.     

Intracellular Replication of Mycoplasmavirus MVL51

The intracellular replication of MVL51, a group L1 mycoplasmavirus, was investigated. The single-stranded parental DNA was found to enter the cell and become converted to double-stranded DNA. This replicated to yield additional double-stranded DNA molecules. The parental viral DNA was found to leave the replication complex and become associated with large molecular weight DNA not involved with viral replication. Progeny viral DNA formed from the double-stranded DNA and an intracellular accumulation of virus chromosome size DNA was observed. The interpretation of this data and a suggested model for the viral replication are discussed and compared to viral DNA replication models for other single-stranded DNA viruses.     

Inhibition of Bacteriophage φX174 DNA Replication in dnaB Mutants of Escherichia coli C

Bacteriophage phiX174 DNA replication was examined in temperature-sensitive dnaB mutants of Escherichia coli C to determine which stages require the participation of the product of this host gene. The conversion of the infecting phage single-stranded DNA to the double-stranded replicative form (parental RF synthesis) is completely inhibited at the nonpermissive temperature (41 C) in two of the three dnaB mutants tested. The efficiency of phage eclipse and of phage DNA penetration of these mutant host cells at 41 C is the same as that of the parent host strain. The defect is most likely in the synthesis of the complementary strand DNA. The semiconservative replication of the double-stranded replicative form DNA (RF replication) is inhibited in all three host mutants after shifting from 30 to 41 C. Late in infection, the rate of progeny single-stranded phage DNA synthesis increases following shifts from 30 to 41 C. Approximately the same amounts of phage DNA and of infectious phage particles are made following the shift to 41 C as in the control left at 30 C. The simplest interpretation of our data is that the product of the host dnaB gene is required for phiX174 parental RF synthesis and RF replication, but is not directly involved in phage single-stranded DNA synthesis once it has begun. The possible significance of the synthesis of parental RF DNA at 41 C in one of the three mutants is discussed.     

The L2 loop peptide of RecA stiffens and restricts base motions of single-stranded DNA similar to the intact protein

The L2 loop in the RecA protein is the catalytic center for DNA strand exchange. Here we investigate the DNA binding properties of the L2 loop peptide using optical spectroscopy with polarized light. Both fluorescence intensity and anisotropy of an etheno-modified poly(dA) increase upon peptide binding, indicate that the base motions of single-stranded DNA are restricted in the complex. In agreement with this conclusion, the peptide-poly(dT) complex exhibits a significant linear dichroism signal. The peptide is also found to modify the structure of double-stranded DNA, but does not denature it. It is inferred that strand separation may not be required for the formation of a joint molecule.     

Copy choice illegitimate DNA recombination

Precise excision of Tn10 and related transposons occurs by recombination between directly repeated 9 bp sequences that flank the transposon. The excision, which is a model for a class of illegitimate DNA recombination events, was stimulated 10(6) times by induction of single-stranded DNA synthesis, occurred during conversion of single-stranded DNA to double-stranded form, and entailed no transfer of physical material from parental to progeny molecules. We conclude that it occurred by copy choice DNA recombination and suggest that other illegitimate recombination events may occur by a similar mechanism.     

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg²⁺-dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg²⁺-independent.     

Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion.

DpnI can cleave fully methylated parental DNA while leaving hemi-methylated DNA intact. Based on this observation, we developed a rapid site-directed mutagenesis method using uracil-containing, double-stranded (ds)DNA templates and DpnI digestion. A 38% mutation efficiency was achieved by DpnI treatment of the mutagenic strand-extension reaction, and it increased to 70%-91% when uracil-containing dsDNA templates were used. This method compares favorably to the most efficient current methods, but is simpler and does not require the use of single-stranded templates or phage vectors.     

Enzymes from Micrococcus luteus involved in the initial steps of excision repair of spontaneous DNA lesions: uracil-DNA-glycosidase and apurinic-endonucleases.

Uracil-DNA-glycosidase that releases free uracil from single-stranded or double-stranded deaminated DNA and poly d(A-U) has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 16,000 and can be separated from uracil-endonuclease and endonucleases (AP-endonucleases) specific for apurinic and apyrimidinic sites. Uracil-DNA-glycosidase does not act on guanine residues opposite uracil in double-stranded DNA and on xanthine in deaminated DNA. The glycosidase generates apyrimidinic sites which can serve as substrate sites for different AP-endonucleases from M. luteus.     

Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA

Ribosome-inactivating proteins (RIPs) are N-glycosylases that remove a specific adenine from the sarcin/ricin loop of the large rRNA in a manner analogous to N-glycosylases that are involved in DNA repair. Some RIPs have been reported to remove adenines from single-stranded DNA and cleave double-stranded supercoiled DNA. The molecular basis for the activity of RIPs on double-stranded DNA is not known. Pokeweed antiviral protein (PAP), a single-chain RIP from Phytolacca americana, cleaves supercoiled DNA into relaxed and linear forms. Double-stranded DNA treated with PAP contains apurinic/apyrimidinic (AP) sites due to the removal of adenine. Using an active-site mutant of PAP (PAPx) which does not depurinate rRNA, we present evidence that double-stranded DNA treated with PAPx does not contain AP sites and is not cleaved. These results demonstrate for the first time that PAP cleaves supercoiled double-stranded DNA using the same active site that is required for depurination of rRNA.     

An auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.     

Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein.

Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an initiator of the replication by inducing a single-stranded cut at the origin of replication (ori). The exact location of the cut induced by the A protein in vivo is determined in this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (iii) primer extensions. It is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, in a region devoid of secondary structures. The A gene has been cloned into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but it cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.     

Studies on the Mechanism of Replication of Adenovirus DNA II. The Nature of Single-Stranded DNA in Replicative Intermediates

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.     

The mechanism of action of the deoxyribonuclease of rat liver chromatin on DNA]

A mechanism of action of DNAase, isolated by chromatin extraction with 0.4 M NaCl, on DNA is described. The enzyme is an endonuclease, it does not require the presence of double-stranded regions in the DNA molecule, does not distinct single-stranded breaks in DNA, and it preferably attacks single-stranded DNA. It hydrolyses DNA for 3'-phosphodiester bonds to octane nucleotides which are resistant to the enzyme activity. The action of the enzyme on DNA does not depend on the position of terminal phosphates. Chromatin DNAase is not specific to DNAs from different origins.     

Interaction of gene 5 protein with DNA

Gene 5 protein bound to both linear and circular single-stranded DNA and saturated the DNA at a protein-to-DNA weight ratio of 7–8. The viscosity of a complex of the protein with single-stranded DNA was initially less than that of the DNA and slowly increased with time suggesting that the complex adopts its final hydrodynamic shape very slowly. This shape change was confirmed by gradient centrifugation. The complex has a more extended structure than DNA alone accounting for its high viscosity and low S value. Gene 5 protein also bound to linear double-stranded DNA though not as strongly as to single-stranded DNA. The protein decreased the transition temperature, T_m, for viscosity loss of double-stranded DNA by 20 °C in 1 and 10 mm salt at a protein-to-DNA ratio of 2.2. At these low ratios there was no decrease in the hyperchromic T_m at 260 nm. At higher ratios of protein to DNA, the hyperchromic T_m was decreased to a constant value and not by a constant amount. Under no conditions was gene 5 protein able to completely separate the complementary strands of double-stranded DNA or to renature denatured DNA.     

The process of infection with bacteriophage φX174: XXXIX. The structure of a DNA form with restricted binding of intercalating dyes observed during synthesis of φX single-stranded DNA

A DNA form with restricted binding of intercalating dyes (propidium iodide or ethidium bromide) has been found in bacteriophage φX-infected cells during the period of single-stranded DNA synthesis. In the electron microscope, this DNA form is seen to be a double-stranded DNA ring with two single-stranded DNA tails protruding from the same portion of the ring; it is composed of a linear φX DNA strand, longer than one φX genome, and a single-stranded ring complementary to φX DNA. Base-pairing of these two tails in partially complementary regions restricts unwinding of the double-stranded DNA ring and consequently intercalation and binding of the dyes. It is postulated that these molecules originate from a previously reported precursor of φX DNA, namely a double-stranded ring with a single-stranded tail, by branch migration.