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D Dressler

Publications and source records attributed to D Dressler.

At least 109 records · Page 6Linked to original sources
PubMed1981

Genome fusion.

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Bacterial Proteins↗
PubMed1980

The rolling circle . capsid complex as an intermediate in phi X DNA replication and viral assembly.

Late in the life cycle of the single-stranded DNA phage phi X, the synthesis of positive strand DNA is coupled to the maturation of progeny virions. DNA synthesis and packaging take place in a replication-assembly complex, which we have purified to homogeneity and characterized. The following conclusions can be drawn: 1. The DNA component of the replication-assembly complex is a rolling circle with a single-stranded DNA tail which is less than or equal to genome length. 2. The major protein component of the replication-assembly complex is an intact viral capsid, as shown by gel analysis of 35S-labeled complexes. As replication proceeds at the DNA growing point, the positive strand tail of the rolling circle is displaced directly into the capsid. In addition to the capsid, the complex contains at least 1 molecule of the phi X gene A nicking protein, which appears to be covalently linked to the DNA. 3. The rolling circle . capsid complex can be purified to homogeneity by taking advantage of its uniform sedimentation velocity (35 S) and its uniform density upon equilibrium centrifugation in CsCl (1.50 g/cc). 4. The replication-assembly complex can be visualized in the electron microscope. An electron-dense particle, which has the dimensions of a viral capsid, is observed attached to a rolling circle at the DNA growing point. 5. Hybridization of specific phi X restriction fragments to the deproteinized, single-stranded tails of intact rolling circles has allowed the use of these replicating intermediates to determine both the origin/terminus and the direction of phi X positive strand DNA synthesis. The ends of the rolling circle tails map in the Hae III restriction Fragment Z6b, at the position on the phi X genome at which the gene A endonuclease is known to cut. This result indicates that this endonuclease participates in the "termination" of each round of synthesis by cutting off unit-length viral genomes. 6. Rolling circle . capsid complexes were also isolated from two other icosahedral, single-stranded DNA phages: G4 and St-1. The rolling circle . capsid complex seen in the case of the single-stranded DNA phages represents the first example of a structure in which DNA synthesis and viral assembly occur in a coupled manner. This tight coordination explains why double-stranded DNA circles are the net product of synthesis early in the viral life cycle while only single-stranded DNA circles are produced later. The single-stranded tails of the rolling circle intermediates are available for conversion to the duplex state at early times, whereas the concentration of preformed capsids later is high enough to bind to all of the replicating molecules and package the emerging positive strand DNA.

Bacteriophage phi X 174↗
PubMed1980

DNA synaptase: an enzyme that fuses DNA molecules at a region of homology.

This paper describes an enzyme from Escherichia coli, and its purification to apparent homogeneity. The protein, which we call "DNA synaptase" and which may be representative of a class of enzymes, fuses double-stranded DNA molecules at a region of homology. In addition, the purified enzyme is able to catalyze the association of single-stranded DNA with homologous duplex DNA. The genome fusion reaction catalyzed by the purified enzyme occurs in the presence of Mg2+, spermidine, and 2-mercaptoethanol and does not require a high-energy cofactor. By bringing two genomes together at a region of homology, DNA synaptase has a property expected for an enzyme that participates in an early step in genetic recombination. However, the synaptase can be recovered from Rec A- cells, and thus it is not yet possible to determine whether this enzyme plays a role in physiological recombination or in another cellular process that involves genome fusion, such as the recombinational repair of damaged DNA.

Bacterial Proteins↗
PubMed1979

dnaG (primase)-dependent origins of DNA replication. Nucleotide sequences of the negative strand initiation sites of bacteriophages St-1, phi K, and alpha 3.

The simplest known origins of DNA replication occur in the single-stranded bacteriophages. In one set of phages, negative strand synthesis is initiated by a single protein, the product of the Escherichia coli replication gene dnaG. Evidently, in these phages--G4, St-1, phi K, and alpha 3--the origin for negative strand synthesis consists of a nucleic acid element capable of direct recognition by the dnaG priming protein. We have located and sequenced the origins of negative strand synthesis in St-1, phi K, and alpha 3, and compared them with the origin sequence previously determined for G4. In each case, the point at which the negative strand is initiated can be identified at the nucleotide level. The data lead to the following conclusions: 1. In all four phages, the negative strand initiation site occurs within an intercistronic region of approximately 135 bases. While in G4, the origin lies between genes specifying the viral coat proteins F and G, the origin is shifted in St-1, phi K, and alpha 3 to a position between coat protein genes G and H. 2. Extensive nucleotide conservation exists at the negative strand origin, but does not extend into the adjacent coding regions. The conserved origin DNA occurs in two regions, 42 and 45 bases long, which are separated by 13 bases of divergent sequence. 3. Correlated with the two stretches of conserved nucleotide sequence are two regions of potential secondary structure. The start point of negative strand synthesis lies just prior to one of these hairpins. Similarities in both primary sequence and secondary structure can be found between the negative strand origins of G4, St-1, phi K, and alpha 3 and the general origin regions of bacteriophage lambda and of E. coli.

Base Sequence↗
PubMed1979

Biochemical assay designed to detect formation of recombination intermediates in vitro.

A biochemical assay that is designed to detect recombination intermediates formed in vitro is described. The assay measures the fusion of two essentially homologous plasmids, one of which is radioactively labeled and the other of which carries several copies of the lac operator. The fusion product is radioactive and can be bound to a nitrocellulose filter by lac repressor. This assay for genome fusion is rapid and readily applicable to the many fractions that result during enzyme purification. The fused product is not destroyed in the assay and may be recovered from the filter for further analysis by electron microscopy. The product is then seen to consist of figure 8 structures that can be cleaved by the restriction enzyme EcoRI to give chi forms, structures similar to those recovered from recombination-proficient cells. It is expected that this assay will be useful in the purification of the "recombinase-type" activity detected in crude cell lysates. To demonstrate this point, the assay was applied to the protein fractions recovered from a molecular sieve column. The results indicate that the fusion activity has an apparent molecular weight of 50,000--100,000.

DNA Restriction Enzymes↗
PubMed1978

Analysis of the phiX DNA replication cycle by electron microscopy.

We have monitored the development of intracellular phiX DNA forms during the course of a virus life cycle that duplicates as closely as possible the normal infection of individual cells by single virions. The viral DNA was isolated in a one-step purification procedure, and quantitative electron microscopy was performed on the samples, resulting in the following conclusions: (i) Early in the life cycle, when the cells accumulate duplex rings, two types of DNA replication intermediates are observed: a rolling circle with a single-stranded tail; and a novel form, a single-stranded circle that is partially duplex. Thus, duplex ring synthesis appears to occur in two asymmetric steps, with positive strand DNA first being processed from the tail of the rolling circle and circularized, before it acts as a template for negative strand synthesis. (ii) Late in the life cycle, as single-stranded circles are synthesized and virus particles are assembled, only one replicating intermediate is observed--the rolling circle with a single-stranded tail. At this stage, the number of rolling circles reaches a level of about 35 per cell. (iii) The net rate of polymerization in the rolling circle intermediates is about 200 nucleotides per sec.

Coliphages↗
PubMed1978

Site-specific initiation of a DNA fragment.

The first step in the replication cycle of the single-stranded DNA phages is the conversion of the infecting positive strand circle to a duplex ring. This event involves the de novo initiation of a negative strand, using the infecting positive strand cycle as a template. The synthesis of the negative strand is in many respects analogous to the formation of a fragment during cellular DNA replication. In this paper we describe the initiation of the negative strand of bacteriophage G4. The data establish that, in vivo, the synthesis of the G4 negative strand is initiated at a specific site, which we have mapped on the 5400-base viral chromosome.

Chromosome Mapping↗
PubMed1978

Site-specific initiation of a DNA fragment: nucleotide sequence of the bacteriophage G4 negative-strand initiation site.

The synthesis of the bacteriophage G4 negative strand is an example of the de novo initiation of a polynucleotide chain. This initiation is performed by the Escherichia coli replication protein dna G which selects a unique site on 5400-base positive-strand template. In this paper we present the nucleotide sequence of the G4 negative-strand initiation site. This is the template element recognized by the dna G priming protein. In conjunction with the sequence of the nascent negative strand, obtained by Bouché, Rowen, and Kornberg [Bouché, J.-P., Rowen, L. & Kornberg, A. (1978) J. Biol. Chem. 253, 765-769], the present data provide a description of a dna G-dependent origin of replication in which one knows the place at which polymerization starts at the nucleotide level.

Base Sequence↗
PubMed1978

In vitro system from Escherichia coli that catalyzes generalized genetic recombination.

This paper reports an in vitro system for studying generalized genetic recombination. The system uses extracts from Escherichia coli as a source of enzymes and plasmid DNA molecules as substrates. Unit-size plasmid DNA rings are converted into genomes fused at a region of DNA homology at a frequency of about 5-10% over a period of hours. That the fused structures are the result of recombination is supported by two lines of evidence. When two partially homologous plasmids of different sizes are used as substrates for the in vitro system, intermediates containing one plasmid of each size are obtained. Furthermore, fused structures are not formed with high efficiency in extracts from recombination-deficient (Rec A(-)) cells.DNA synthesis does not appear to be required for the formation of the recombination intermediates; it is possible to omit DNA precursors from the reaction mixture and, furthermore, to develop the fused structures even in the presence of chaintermininating dideoxynucleoside triphosphates. The structures formed in vitro have the basic properties of recombination intermediates previously recovered from intact cells. That is, two genomes are demonstrably fused at a region of homology. However, in one way the molecules formed in vitro have a property less frequently observed in vivo-the fused genomes often appear to be connected over an extended region of homology ranging up to several hundred base pairs in length. This extended region of pairing may indicate the presence of two crossover connections very close together and, as will be discussed, may provide an insight into the mechanism by which the recombination intermediate is formed.

Crossing Over, Genetic↗
PubMed1977

On the mechanism of genetic recombination: the maturation of recombination intermediates.

DNA molecules of the plasmid ColEl are normally recovered from wild-type cells as a set of monomer- and multimer-size rings. The data of this paper show that the multimer-size species are a product of genetic recombination. Multimer rings do not arise after transfection of purified monomers into bacterial host cells lacking a functional recA recombination system. Analogously, purified dimers, trimers, and tetramers, transfected into recA- cells, can replicate, but are constrained to remain in those conformations. Only upon transfection into rec+ cells can they regenerate the full spectrum of monomer- and multimer-size species. In this paper we trace the flow of genetic information from the monomer to the multimer state and back again under the guidance of the recA recombination system. The formation of multimer-size DNA rings is discussed as a natural consequence of the maturation of a Holliday recombination intermediate formed between two monomer plasmid genomes.

Chromosomes↗
PubMed1976

On the mechanism of genetic recombination: electron microscopic observation of recombination intermediates.

This paper deals with the nature of recombination intermediates. Using the electron microscope to study the DNA of the plasmid colicin E1, we have observed more than 800 molecules that appear to represent intermediates in the process of recombination. Specifically, after isolating colicin DNA and linearizing it with the restriction enzyme EcoRI, we find crossed molecules with twice the normal colicin DNA content. These forms consist of two genome-length elements held together at a region of DNA homology. The molecules can be recovered from wild type and Rec B-C host cells but are not present among the colicin DNA forms isolated from recombination-deficient Rec A cells. We have termed the experimentally observed molecules "chi forms" and believe that they represent the recombination intermediate of the Holliday model.

Colicins↗
PubMed1976

[Gout, pseudo-gout or hyperlipoproteinemia? (author's transl)].

A number of patients with aseptic necroses of bone and hyperlipoproteinemia of types II and IV after Fredrickson are reported on. More than one half of them had hyperuricemia without symptoms of gout. The relationship between fat-, purine- and carbohydrate-metabolism are discussed. The clinical pictures of hyperuricemia, gout and hyperlipoproteinemia are shown to resemble each other. Differential diagnosis, particularly as related to aseptic necroses of bone, is discussed in detail.

Bone Diseases↗