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H Stahl

Publications and source records attributed to H Stahl.

At least 37 records · Page 2Linked to original sources
PubMed1993

p53-catalyzed annealing of complementary single-stranded nucleic acids.

p53 has been reported to inhibit the DNA helicase intrinsic to simian virus 40 large tumor antigen (T antigen). We found that inhibition is not restricted to T antigen, but also affects several other DNA and RNA helicases. Complexing of the helicases by the p53 protein as a possible inactivation mechanism could be excluded. Instead, the anti-helicase activity can be explained by our finding that p53 binds with high affinity to single-stranded nucleic acids and has a strong DNA.DNA and RNA.RNA annealing activity. We could also show that p53 is able to alter the secondary structure of RNA and/or to influence dynamic RNA-RNA interactions. These results, and the fact that the affinity of p53 to RNA is about one order of magnitude higher than to single-stranded DNA, imply an RNA-specific function of p53 in vivo.

Animals↗
PubMed1993

Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.

Alanine↗
PubMed1992

The interaction of SV40 large T antigen with unspecific double-stranded DNA: an electron microscopic study.

T antigen, an early protein encoded by simian virus 40 (SV40), is a specific DNA-binding protein with high affinity for elements in the viral origin of replication where it forms a double-hexameric complex as a prerequisite for DNA untwisting and, in the presence of ATP hydrolysis, for DNA unwinding. Like other specific DNA-binding proteins, T antigen also associates with DNA strands of random sequence albeit at reduced affinity. In addition, T antigen is able to unwind unspecific DNA sequences starting from internal binding sites. This property could be a step in the pathway leading to the chromosomal rearrangements that are frequently observed in SV40-transformed cells. This possibility prompted us to investigate the binding of T antigen to unspecific DNA using electron microscopy. We observed that the protein binds randomly to many unspecific DNA sites excluding a preference for particular DNA sequences or structural features. Addition of ATP to the binding buffer induces the formation of oligomeric, possibly hexameric, T antigen complexes that frequently align to form long arrays of DNA-bound protein. Magnesium salts induce the formation of tightly packed T antigen aggregates which bind to DNA to form many DNA branches and loops that emanate from the aggregated protein core. Upon ATP hydrolysis, aggregated T antigen catalyzes the unwinding of DNA duplices.

Adenosine Triphosphate↗
PubMed1992

Simian virus 40 T-antigen DNA helicase is a hexamer which forms a binary complex during bidirectional unwinding from the viral origin of DNA replication.

The role of simian virus 40 (SV40) large tumor antigen (T antigen) as a DNA helicase at the replication fork was studied. We found that a T-antigen hexamer complex acts during the unidirectional unwinding of appropriate DNA substrates and is localized directly in the center of the fork, contacting the adjacent double strand as well as the emerging single strands. When bidirectional DNA unwinding, initiated at the viral origin of DNA replication, was analyzed, a larger T-antigen complex that is simultaneously active at both branch points of an unwinding bubble was observed. The size and shape of this helicase complex imply that the T-antigen dodecamer complex, assembled at the origin and active in the localized melting of duplex DNA, is subsequently also used to continue DNA unwinding bidirectionally. Then, however, the dodecamer complex does not split into two hexamer subunits that track along the DNA; rather, the DNA is threaded through the intact complex, with the concomitant extrusion of single-stranded loops.

Antigens, Polyomavirus Transforming↗
PubMed1991

Simian-virus-40 large-T-antigen-catalyzed DNA and RNA unwinding reactions.

Simian virus 40 large T antigen is a helicase separating the complementary strands of double-stranded DNA in the presence of hydrolyzable ATP and of double-stranded RNA in the presence of non-ATP nucleotides (GTP, CTP or UTP). We have constructed partially single-stranded nucleic acid substrates consisting of RNA or DNA strands hydrogen bonded to either RNA or DNA complements. We found that ATP is utilized as a cofactor for the T-antigen-catalyzed unwinding reaction when the substrates contain overhanging single-stranded DNA, regardless of whether the double-stranded region is DNA or hybrid DNA.RNA. Conversely, non-ATP nucleotides are used when the overhanging single strand is RNA. Based on these and additional findings, we propose that the bound nucleic acid induces a conformational change in T antigen resulting in a proper orientation of both nucleic acid and nucleotide relative to the active center of the ATPase/helicase domain of the enzyme. The implications of our conclusion for the roles which T antigen may play in vivo are discussed.

Antibodies, Monoclonal↗
PubMed1991

PIF1: a DNA helicase in yeast mitochondria.

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix-localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single-stranded (ss) DNA-dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5' to 3' direction with respect to the ss DNA on which it binds first.

Adenosine Triphosphatases↗
PubMed1990

Renaturation and DNA looping promoted by the SV40 large tumour antigen.

Simian virus 40 large tumour antigen (T antigen) is shown to catalyse the formation of duplex DNA from complementary strands in specific conditions. The activity is dependent on an excess of unspecific double-stranded DNA and seems not to function by T antigen mediated destabilization of secondary structure. Rather, protein-protein contacts between T antigen molecules appear to be involved. Protein-protein interactions between T antigen molecules bound to physically separated DNA sites are also demonstrated by the formation of specific DNA loops and by cyclization of DNA molecules with 3'-extended single-stranded ends where T antigen specifically binds to the single-stranded/double-stranded junctions. The relevance of these properties for T antigen functions in DNA replication, transcription and(or) recombination is discussed.

Antigens, Polyomavirus Transforming↗
PubMed1990

[Characteristics of the relative humidity and temperature in the inspiratory part of the Dräger circle system and their influence on the function of the ciliary epithelium].

Changes in relative humidity and temperature of the anaesthetic gases were measured in the inspiratory limb of the Dräger circle system next to the carbon dioxide absorber in 29 patients requiring ENT surgery under general anaesthesia. Immediately following intubation and prior to extubation, nasal and tracheal cytologic samples were taken with a brush technique and ciliary beat frequency was determined. At a fresh gas flow of 6 l/min, relative humidity increased from 57.6 +/- 1.5 to 62.5 +/- 1.8% (p less than 0.05) after 110 minutes. Temperature increased continuously from 21.96 +/- 0.97 degrees C to 23.83 +/- 0.48 degrees C after 200 minutes. The number of vital ciliated cells in the tracheal samples decreased from 24.4% following induction of anaesthesia to 6% at the end of anaesthesia (p less than 0.05), and from 35.7% to 26.8% (p less than 0.05) in the nasal samples. Ciliary beat frequency remained unchanged at the end of anaesthesia as compared to control in tracheal as well as in nasal samples. It is concluded that the output of relative humidity and temperature in the circle system is not sufficient to prevent broncho-epithelial damage. Ciliary beat automaticity appears to behave according to an all or nothing principle.

Adult↗
PubMed1989

[Observations on science and religion].

Our view of the universe used to be primed by religious terms. Our perception of the world has changed fundamentally under the influence of natural sciences. Today religion faces the difficult task to integrate scientific knowledge into its teaching.

Humans↗
PubMed1989

RNA unwinding activity of SV40 large T antigen.

Large T antigen, the regulatory protein encoded by simian virus 40, has DNA helicase activity and unwinds double-stranded DNA at the expense of ATP. T antigen also functions as an RNA helicase separating duplex regions in partially double-stranded RNA substrates. Surprisingly, T antigen RNA helicase activity requires UTP, CTP, or GTP as a cofactor, whereas ATP is an inefficient energy source for the RNA unwinding reaction. Accordingly, T antigen has both an intrinsic non-ATP NTPase activity that is stimulated by single-stranded RNA and an ATPase activity stimulated by single-stranded DNA. Thus, it appears that the bound nucleotide determines whether T antigen acts as an RNA helicase or as a DNA helicase.

Antigens, Polyomavirus Transforming↗
PubMed1989

RNA helicase activity associated with the human p68 protein.

It has been proposed that p68, a nuclear protein of relative molecular mass 68,000, functions in the regulation of cell growth and division. A complementary DNA analysis of the protein has revealed extensive amino-acid sequence homology to the products of a set of genes recently identified in organisms as diverse as Escherichia coli and man, which include the eukaryotic translation initiation factor elF-4A. The protein products of the new gene family have several motifs in common which are thought to be involved in nucleic acid unwinding. As yet, however, only elF-4A, through its effect on RNA, has been shown to possess unwinding activity. Here we report that purified p68 also exhibits RNA-dependent ATPase activity and functions as an RNA helicase in vitro. The protein was first identified by its specific immunological cross reaction with the simian virus 40 large T antigen, the transforming protein of a small DNA tumour virus. Surprisingly, T antigen also has an RNA-unwinding activity: the homology between the two polypeptides, although confined to only a small region resembling the epitope of the cross-reacting antibody (PAb204), should therefore be of functional significance. Furthermore, the RNA-unwinding activity may be involved in the growth-regulating functions of both proteins.

Adenosine Triphosphatases↗
PubMed1989

DNA binding properties and replication activity of the T antigen related D2 phosphoprotein.

According to earlier genetic experiments, a region within the N-terminal 50-100 amino acids may be important for the replication function of T antigen, the initiator protein of simian virus 40 (SV40). We have investigated this possibility using the T antigen related D2 protein in several biochemical assay systems. D2 protein, a phosphoprotein coded for by the adeno-SV40 hybrid virus Ad2+D2, shares its 594 C-terminal amino acids with authentic T antigen and its 104 N-terminal amino acids with an adenovirus structural protein. We confirmed earlier studies showing that D2 protein appeared to bind well to specific binding sites in the SV40 origin of replication. We found, however, that D2 protein was rather inefficient, inducing the unwinding of the double-stranded origin region, and was much less active than authentic T antigen as an initiator of in vitro SV40 DNA replication. We interpret these findings to indicate that D2 protein molecules associate with the origin to form an aberrant complex that is quite inefficient, inducing DNA unwinding and the establishment of replication forks. The possibility that the N-terminus may be required for an optimal arrangement of T antigen at the origin was supported by results of dephosphorylation studies. Dephosphorylation of N-terminal phosphoamino acids had significant effects on the stability of D2 protein-origin complexes.

Antigens, Viral, Tumor↗
PubMed1989

Sequence independent duplex DNA opening reaction catalysed by SV40 large tumor antigen.

Simian virus 40 (SV40) large tumor antigen (T antigen) is mainly localized in the nucleus where it exhibits two biochemical properties: DNA binding and helicase activity. Both activities are necessary for viral DNA replication and may also enable T antigen to modulate cellular growth. Here we present biochemical and electron microscopic evidence that the helicase activity can start at internal sites of fully double-stranded DNA molecules not containing the SV40 origin or replication. Using T antigen specific monoclonal antibodies, this unwinding reaction can be biochemically divided in an initiation (duplex opening) and a propagation step. The duplex opening reaction (as well as the propagation step) does not depend on a specific DNA sequence or secondary structure. In addition, we have found that T antigen forms an ATP dependent nucleoprotein complex at double-stranded DNA, which may be an essential step for the sequence independent duplex DNA opening reaction.

Antibodies, Monoclonal↗
PubMed1988

Protein-DNA interactions at the simian virus 40 origin of replication.

Simian Virus 40 (SV40)-encoded large T antigen has an intrinsic ATP-dependent DNA-unwinding activity which is necessary for an early step in the activation of the viral origin of replication. Isolated T antigen unwinds any double-stranded DNA, regardless of whether it is linear or circularly closed. However, initiation of DNA replication depends on an intact origin of replication, and even minor deviations from the wild-type origin sequence abolish the template activity of an origin-bearing plasmid. This discrepancy suggests that T antigen may not be sufficient for origin activation and that other, probably cellular, functions are involved. We have isolated a cellular protein, the LOB protein, which specifically interacts with the AT-rich region of the SV40 origin and which induces a pronounced bending of the bound DNA.

Antigens, Polyomavirus Transforming↗
PubMed1988

Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements.

The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity. The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates. The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C. For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction. The minimum length of the single-stranded tail was determined to be less than 5 nucleotides. Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction. This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase. Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome. The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.

Antigens, Polyomavirus Transforming↗