Electron microscopy of a dna helix destabilizing protein crystal (gp32*i)

1978 ◽  
Vol 36 (2) ◽  
pp. 178-179 ◽  
Author(s):  
Wah Chiu ◽  
Junko Hosoda
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Dna Replication ◽  
Bacteriophage T4 ◽  
Polystyrene Latex ◽  
Protein Crystal ◽  
Low Salt ◽  
Leading Strand ◽  
Gene 32

Gp 32 is a protein coded by gene 32 in bacteriophage T4. It is essential for DNA replication, recombination and repair. Gp32*I with molecular weight of 27,000 was obtained by proteolytic removal of 8000-dalton peptide from COOH-terminal of gp32 (Hosoda et al. to be published). It is a stronger helix destabilizer than gp32, and has been found to be able to replace gp32 in constructing an in vitro DNA replication apparatus, which is active in the leading strand synthesis.Thin gp32*I crystal was formed under a low salt condition in the absence of glycerol. Figure 1 shows a typical image of the crystal embedded in 1% glucose taken at low magnification. The optical diffractogram in the insert confirms the crystallinity, though no visible feature is seen in the micrograph due to the low magnification power. By using the tungsten shadowing technique, the thickness of the crystal was measured, along with polystyrene latex spheres as a standard, to be between 85 to 400 Å.

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex

1992 ◽  
Vol 12 (1) ◽  
pp. 155-163 ◽  
Author(s):  
K Fien ◽  
B Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Factor C ◽  
Dna Polymerase Delta ◽  
Replication Complex ◽  
Leading Strand ◽  
Factor C ◽  
Sv40 Dna

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

scholarly journals Polymerization of actin by positively charged liposomes.

1988 ◽  
Vol 106 (4) ◽  
pp. 1221-1227 ◽  
Author(s):  
A Laliberte ◽  
C Gicquaud
Keyword(s):  
Electron Microscopy ◽  
Direct Contact ◽  
Atp Hydrolysis ◽  
Actin Assembly ◽  
Liposome Membrane ◽  
Low Salt ◽  
Spectrofluorimetric Analysis ◽  
Positively Charged

By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.

Autogenous translational repression of bacteriophage T4 gene 32 expression in vitro

1978 ◽  
Vol 126 (1) ◽  
pp. 73-90 ◽  
Author(s):  
Geneviève Lemaire ◽  
Larry Gold ◽  
M. Yarus
Keyword(s):  
Bacteriophage T4 ◽  
Translational Repression ◽  
Gene 32

scholarly journals Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

2019 ◽  
Vol 116 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Glen E. Cronan ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Chromosomal Dna ◽  
E Coli ◽  
Okazaki Fragments ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

scholarly journals Studies on the Recombination Genes of Bacteriophage T4: Suppression of uvsX and uvsY Mutations by uvsW Mutations

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 219-227
Author(s):  
Tetsuro Yonesaki ◽  
Teiichi Minagawa
Keyword(s):  
Molecular Weight ◽  
Dna Synthesis ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
Uv Resistance ◽  
Small Plaque ◽  
New Mutation ◽  
Single Stranded Dna ◽  
Large Plaque ◽  
Gene 32

ABSTRACT Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42° and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41° the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30° it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41° was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.

scholarly journals Recombination and Pol ζ Rescue Defective DNA Replication upon Impaired CMG Helicase—Pol ε Interaction

2020 ◽  
Vol 21 (24) ◽  
pp. 9484
Author(s):  
Milena Denkiewicz-Kruk ◽  
Malgorzata Jedrychowska ◽  
Shizuko Endo ◽  
Hiroyuki Araki ◽  
Piotr Jonczyk ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Synthetic Lethality ◽  
Replication Initiation ◽  
Recombination Rates ◽  
Dna Polymerase Epsilon ◽  
Dna Replication Initiation ◽  
Leading Strand

The CMG complex (Cdc45, Mcm2–7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals The cercarial glycocalyx of Schistosoma mansoni.

1985 ◽  
Vol 100 (5) ◽  
pp. 1423-1434 ◽  
Author(s):  
J C Samuelson ◽  
J P Caulfield
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Schistosoma Mansoni ◽  
High Molecular Weight ◽  
Membrane Surface ◽  
Periodate Oxidation ◽  
Apparent Molecular Weight ◽  
Ruthenium Red ◽  
Unit Membrane

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.

Immunolocalization and characterization of the low molecular weight antigen (4–5 kDa) ofToxoplasma gondiithat elicits an early IgM response upon primary infection

Parasitology ◽  
1994 ◽  
Vol 108 (2) ◽  
pp. 139-145 ◽  
Author(s):  
S. Tomavo ◽  
G. Couvreur ◽  
M. A. Leriche ◽  
A. Sadak ◽  
A. Achbarou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Primary Infection ◽  
Low Molecular Weight ◽  
Immuno Electron Microscopy ◽  
Surface Localization

SUMMARYA striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4–5 kDa. Two different monoclonal antibodies directed against the 4–5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the lowMrantigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4–5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability betweenToxoplasma gondiitachyzoites grownin vivoandin vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

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