Replication of mycoplasmavirus MVL51: I. Replicative intermediates.

The intracellular replication of the single stranded DNA of the non-lytic bullet-shaped Group L1 mycoplasmavirus, MVL51, has been shown to involve three virus specific DNAs: RFI, RFII and SS. The relative sedimentation rates and ethidium bromide CsCl gradient analysis show that RFI is covalently closed circular double stranded DNA and RFII is a nicked form of RFI. SS is circular single stranded progeny viral DNA. RFI and RFII serve as precursors for the synthesis of progeny SS.     

Characterization of an Acholeplasma laidlawii variant with a REP- phenotype.

An Acholeplasma laidlawii variant has been isolated that has a REP- phenotype. The properties of this variant, relative to parental cells, are: (i) it exhibits no change in cell growth kinetics; (ii) it does not propagate single-stranded deoxyribonucleic acid (DNA) mycoplasmaviruses but does propagate double-stranded DNA mycoplasmaviruses; (iii) it converts parental circular single-stranded mycoplasmavirus DNA to double-stranded replicative forms that are not replicated further; (iv) it exhibits no change in host modification and restriction; and (v) it has an increased ultraviolet light sensitivity. The REP- isolate is the first stable mycoplasma variant to which a physiological defect has been attributed.     

Characterization of mycoplasmavirus MVL2 DNA.

The size and molecular configuration of mycoplasmavirus MVL2 DNA are presented. The DNA was shown to be a covalently closed circular duplex molecule with a molecular weight of 7.4 X 10(6). In sucrose gradients at neutral pH, the form I and form II DNA molecules have sedimentation values of approximately 29S and 23S, respectively. Under alkaline conditions, the form I and form II have S values of 70 and 20, respectively.     

Structural and biological properties of mycoplasmavirus MVL3: an unusual virus-procaryote interaction.

The kinetics of adsorption and growth of mycoplasmavirus MVL3 in Acholeplasma laidlawii 1305/68 host cells have been studied with one-step growth, premature lysis, and single-burst experiments. The virus was found to kill infected host cells. Virus release starts 90 min after infection and continues for about 10 to 15 h. Hence, virus production is unlike the classical lytic bacteriophages and instead resembles nonlytic cytocidal animal viruses. Structural details of the virus are described, and the molecular weight of the viral linear DNA has beenfound to be 26 x 10(6).     

Virus and host cell DNA syntheses during infection of Acholeplasma laidlawii by MVL3, a nonlytic cytocidal mycoplasmavirus.

The replication of mycoplasmavirus MVL3 in Acholeplasma laidlawii K2 host cells was studied by analysis of infected-cell lysates using sedimentation in sucrose gradients and DNA-DNA hybridization. Viral DNA replication was found to involve intermediates sedimenting faster than free viral DNA, which is a linear, double-stranded molecule of about 26 x 10(6) daltons. After the shutdown of cellular DNA synthesis, viral DNA synthesis continued for many hours. The fate of cellular and parental viral DNAs was examined.     

Properties of a persistent viral infection: possible lysogeny by an enveloped nonlytic mycoplasmavirus.

MVL2, an enveloped double-stranded DNA mycoplasmavirus, causes a nonlytic infection of Acholeplasma laidlawii leading to the establishment of a persistent infection. Persistently infected clones were found to be resistant to superinfection by homologous virus, but could be infected by heterologous virus. Cells in a persistently infected culture had the potential to produce virus and transmitted this potential as a stable heritable trait. Mitomycin C and UV light induced an increase in infectious centers in persistently infected cultures.     

The DNA binding properties of the MutL protein isolated from Escherichia coli.

The mutL gene of Escherichia coli, which is involved in the repair of mispaired and unpaired nucleotides in DNA, has been independently cloned and the gene product purified. In addition to restoring methyl-directed DNA repair in extracts prepared from mutL strains, the purified MutL protein binds to both double and single stranded DNA. The affinity constant of MutL for unmethylated single stranded DNA was twice that of its affinity constant for methylated single stranded DNA and methylated or unmethylated double stranded DNA. The binding of MutL to double stranded DNA was not affected by the pattern of DNA methylation or the presence of a MutHLS-repairable lesion.     

Growth of an enveloped mycoplasmavirus and establishment of a carrier state.

The growth of an enveloped DNA-containing mycoplasmavirus (MVL2 obtained from R.N. Gourlay) has studied, by using the indicator host Acholeplasma laidlawii strain JA1. From virus one-step growth curves, artificial lysis experiments, and infected cell growth curves, it was found that virus infection is nonlytic. Newly infected cells grow slower and are osmotically more stable than uninfected cells. However, 4 to 6 h after infection, the cells reach a carrier state in which cell growth rate and osmotic fragility are indistinguishable from uninfected cells. Carrier cultures contain free virus. Every carrier culture cell gives rise to either a clone of carrier cells or a clone of MVL2-resistant cells.     

Host cell and ultraviolet reactivation of ultraviolet-irradiated Mycoplasmaviruses.

The mycoplasma Acholeplasma laidlawii was shown to have mechanisms for both host cell and ultraviolet (UV) reactivation of UV-irradiated mycoplasmaviruses. Host cell reactivation was examined by comparing the survival abilities of UV-irradiated double-stranded deoxyribonucleic acid mycoplasmavirus plated on both untreated and on acriflavine-treated cells. Acriflavine treatment inhibited cell exision repair. Decreased survival on the acriflavine-treated cells demonstrated host cell reactivation. UV reactivation was studied by comparing the survival of UV-irradiated virus plated on untreated cells with its survival on cells that received a small UV dose before plating. The UV-irradiated cells gave increased virus survival, showing UV reactivation. Similar experiments with a single-stranded deoxyribonucleic acid mycoplasmavirus showed that this virus could be UV reactivated, but not host cell reactivated.     

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.     

Site directed in-vitro assembly of nucleosomes.

An in-vitro system is described allowing for the assembly of nucleosomes on preselected sites of a cloned tRNA gene. The system consists of a soluble nucleoprotein fraction, a histone source, and circular DNA containing a single stranded stretch (sssDNA). Nucleosomes assemble on the sssDNA, if the three components are incubated as a highly concentrated solution in the presence of the four deoxyribonucleotidetriphosphates and of ATP. The single stranded stretch is rendered double stranded during incubation. The middle axis of one assembled nucleosome always coincides roughly with the midpoint of the original single stranded DNA stretch.     

Structure of the DNA binding domain of E. coli SSB bound to ssDNA

The structure of the homotetrameric DNA binding domain of the single stranded DNA binding protein from Escherichia coli (Eco SSB) bound to two 35-mer single stranded DNAs was determined to a resolution of 2.8 A. This structure describes the vast network of interactions that results in the extensive wrapping of single stranded DNA around the SSB tetramer and suggests a structural basis for its various binding modes.     

Construction of a 42 base pair double stranded DNA microcircle.

A procedure for the construction of double stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. Starting with two synthetic oligonucleotides which are able to form dumbbell shaped structures, two subsequent ligation reactions yield a microcircle of double stranded DNA of 42 base pairs. This is by far the smallest circle of double stranded DNA yet described. These microcircles can be constructed in quantities required for high resolution structural analyses such as X-ray crystallography and NMR spectroscopy.     

RAD50 protein of S.cerevisiae exhibits ATP-dependent DNA binding.

RAD50 function of Saccharomyces cerevisiae is required during vegetative growth for recombinational repair of DNA double strand breaks, and during meiosis for initiation of meiotic recombination and formation of synaptonemal complex. RAD50 encodes a 153 kDa polypeptide which includes an amino-terminal ATP binding domain essential for function and two long heptad repeat regions. We show below that RAD50 protein purified from yeast exhibits ATP-dependent binding to double stranded DNA. Physical properties of the purified protein are also described. Models for RAD50 function in vivo are discussed.     

Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors.

For the isolation of single stranded plasmid DNA, various E. coli and E. coli-Streptomyces shuttle plasmids were equipped with the phage f1 replication origin. The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used. In contrast, the transformation of Streptomyces was 10 to 100 times more efficient when an integration vector was in the single stranded form as opposed to the double stranded form. Streptomyces viridochromogenes was transformed by single stranded DNA integration vectors in order to replace the pat by the tsr gene and generate mutants unable to synthesize phosphinothricin-tripeptide (PTT).     

Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions.

Binding of the 58 kDa monomer and 44 kDa alpha beta dimer forms of terminal deoxynucleotidyl transferase to double stranded DNA was demonstrated by gel retardation and tryptophan fluorescence quenching. The dissociation constants and cooperativity parameters were similar to those that have been determined for binding of these two forms of terminal transferase to single stranded DNA. However, the double stranded DNA binding site size of 10 nucleotides was half the size expected. The efficacy of blunt ended DNA as an initiator in the polymerization reaction catalyzed by terminal transferase was demonstrated by radiometric assays and product analyses on agarose gels. The initial reaction kinetics indicated that dGTP but not dATP was added efficiently to a blunt double stranded DNA 3' end. These results are correlated with current models for in vivo terminal transferase function.     

The binding of RecA protein to duplex DNA molecules is directional and is promoted by a single stranded region.

RecA protein from E. coli binds more strongly to single stranded DNA than to duplex molecules. Using duplex DNA that contains single stranded gaps, we have studied the protection by RecA protein at various concentrations, of restriction sites as a function of their distance from the single stranded region. We show that the binding of RecA protein, initiated in the single stranded region, extends progressively along the adjoining duplex in the 5' to 3' direction with respect to the single stranded region. The strand exchange reaction is known to proceed in the same direction.     

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.     

DNA-methylase from regenerating rat liver: purification and characterisation.

DNA methylase has been purified 660-fold from nuclei from regenerating rat liver. The enzyme is able to methylate single stranded (ss) and double stranded (ds) DNA, the only reaction product being 5-methylcytosine. Previously unmethylated double stranded DNA from prokaryotes (M.luteus) as well as from eukaryotes (Ascaris suis) can serve as substrates. The synthetic copolymers (dG-dC)n . (dC-dG)n and (dG,dC)n are also methylated. While SV40 DNA is almost not methylated, PM2 DNA is a good substrate even in the supercoiled form. The enzyme methylates 1 in 17 bases in heterologous M.luteus DNA, but only 1 in 590 in homologous rat liver DNA. The high methylation level of M.luteus DNA, an analysis of the methylated pyrimidine isostichs and a preliminary dinucleotide analysis suggest that all the CpGs in a DNA can be methylated.