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R Sternglanz

Publications and source records attributed to R Sternglanz.

70 records · Page 4Linked to original sources
PubMed1984

DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication.

A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-microns plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.

Cell Cycle↗
PubMed1982

Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes.

Escherichia coli deletion mutants lacking DNA topoisomerase I have been identified previously and shown to grow at a normal rate. We show that such strains grow normally only because of spontaneously arising mutations that compensate for the topoisomerase I defect. Several of these compensatory mutations have been found to map at or near the genes encoding DNA gyrase, gyrA and gyrB. DNA gyrase assays of crude extracts show that strains carrying the mutations have lower gyrase activity. Thus the mutations are in the gyrase structural genes or in nearby regulatory sequences. These results, in conjunction with DNA supercoiling measurements of others, indicate that in vivo DNA superhelicity is a result of a balance between topoisomerase I and gyrase activities. An excess of negative supercoils due to an absence of topoisomerase I is deleterious to the cell, but a moderate gyrase deficiency is not harmful.

Chromosome Deletion↗
PubMed1981

Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition.

Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and beta-galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency of Tn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in Salmonella typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.

Chromosomes, Bacterial↗
PubMed1979

Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli.

A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc. Natl. Acad. Sci. U.S.A. 74:3485-3489, 1977). At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures. This suggests that the mutant may be among the most ligase-deficient strains yet characterized.

Chromosome Mapping↗
PubMed1978

Ionization and divalent cation dissociation constants of nalidixic and oxolinic acids.

The ionization constants and some divalent cation dissociation constants of nalidixic and oxolinic acids, both specific inhibitors of bacterial DNA replication, have been determined. The carboxylic pKa' values are 6.1 and 6.9 at 25 degrees for nalidixic and oxolinic acids, respectively. These values indicate that intramolecular hydrogen-bonding stabilizes the un-ionized form of these compounds in aqueous solution. Both compounds bind divalent cations; the divalent cation dissociation constants for oxolinic acid are somewhat smaller that those for nalidixic acid. We suggest that both compounds may act by forming a complex in situ with a divalent cation in a metalloprotein involved in DNA replication. The evidence that both drugs inhibit at the same target site is briefly reviewed.

Cations, Divalent↗
PubMed1977

Recent excitement in the DNA replication problem.

It is now possible to reproduce most of the reactions involved in DNA replication using prokaryotic enzymes in vitro. Such systems have revealed that DNA replication is a complex process depending on a relatively large number of proteins, and that nucleoside triphosphate hydrolysis energy is used at several discrete steps. Much of the complexity of DNA replication may arise from the need for extreme copying fidelity.

Adenosine Triphosphate↗
PubMed1976

Evidence that both growing DNA chains at a replication fork are synthesized discontinuously.

Escherichia coli, Bacillus subtilis, and T7-infected E. coli have been labeled with short pulses of [3H] thymidine, and the labeled DNA has been examined by sedimentation in alkaline sucrose. In all three systems, the great majority of the DNA labeled by a short pulse is found in the form of small DNA chains of 10S, the so-called Okazaki pieces. The B. subtilis and T7 nascent DNA fragments hybridize with equal efficiency to the separated strands of B. subtilis and T7 DNA, respectively. The results suggest that both growing DNA chains at a given replication fork are synthesized discontinuously in the case of E. coli, B. subtilis, and T7. We have found that the method used to terminate the pulse affects the size distribution of the labeled DNA; some methods allow joining of nascent DNA fragments after termination of the pulse. Previous reports of discontinuous DNA synthesis on only one growing DNA chain and continuous synthesis on the other DNA chain are probably due to preferential joining of Okazaki pieces on the DNA chain growing in the overall 5' leads to 3' direction.

Bacillus subtilis↗
PubMed1973

Studies on the mechanism of action of nalidixic acid.

With three independent techniques (absorption spectrophotometry, measurement of the deoxyribonucleic acid [DNA] melting temperature, and equilibrium dialysis), no evidence has been found for the binding of nalidixic acid to purified DNA. Also, no evidence has been found to support the hypothesis that nalidixic acid is permanently modified to a new, active compound by the bacterial cell. By using an in vitro DNA replication system developed by Bonhoeffer and colleagues, soluble extracts from nalidixic acid-sensitive cells have been shown to confer nalidixic acid sensitivity on the DNA synthesis of lysates from nalidixic acid-resistant cells. The activity in the extracts is only present in sensitive cells and is nondialyzable and heat sensitive. Finally, two known nalidixic acid-resistant mutants of Escherichia coli, mapping at nal A and nal B, respectively, have been tested to determine whether either of them is a transport mutant. It has been shown that nal B(r) is a transport mutant whereas nal A(r) is not.

DNA Replication↗
PubMed1972

Effect of nalidixic acid on the growth of deoxyribonucleic acid bacteriophages.

The effect of nalidixic acid on the growth of various deoxyribonucleic acid (DNA) bacteriophages has been investigated by one-step growth experiments. The Escherichia coli bacteriophages T5, lambda, T7 and phiR are strongly inhibited by nalidixic acid, whereas T4 and T2 are only partially inhibited. The Bacillus subtilis bacteriophages SP82, SP50, and phi29 are relatively unaffected by nalidixic acid. There is no correlation between those bacteriophages which can grow in the presence of nalidixic acid and the presence of an unusual base in the phage DNA.

Bacillus subtilis↗
PubMedDate not supplied

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA.

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).

DNA Replication↗