Characterization of DNA synthesis at a restrictive temperature in the temperature-sensitive mutants, tsFT5 cells, that belong to the complementation group of ts85 cells containing a thermolabile ubiquitin-activating enzyme E1. Involvement of the ubiquitin-conjugating system in DNA replication

ABSTRACT Previous analyses of randomly generated, temperature-sensitive vaccinia virus mutants led to the mapping of DNA synthesis negative complementation groups to the B1R, D4R, D5R, and E9L genes. Evidence from the yeast two-hybrid system that the D4R and D5R proteins can interact with the A20R protein suggested that A20R was also involved in DNA replication. We found that the A20R gene was transcribed early after infection, consistent with such a role. To investigate the function of the A20R protein, targeted mutations were made by substituting alanines for charged amino acids occurring in 11 different clusters. Four mutants were not isolated, suggesting that they were lethal, two mutants exhibited no temperature sensitivity, two mutants exhibited partial temperature sensitivity, and two mutants formed no plaques or infectious virus at 39°C. The two mutants with stringent phenotypes were further characterized. Temperature shift-up experiments indicated that the crucial period was between 6 and 12 h after infection, making it unlikely that the defect was in virus entry, early gene expression, or a late stage of virus assembly. Similar patterns of metabolically labeled viral early proteins were detected at permissive and nonpermissive temperatures, but one mutant showed an absence of late proteins under the latter conditions. Moreover, no viral DNA synthesis was detected when cells were infected with either stringent mutant at 39°C. The previous yeast two-hybrid analysis together with the present characterization of A20R temperature-sensitive mutants suggested that the A20R, D4R, and D5R proteins are components of a multiprotein DNA replication complex.

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Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4594-4601.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4594-4601

Author(s):

J J Dermody ◽

B E Wojcik ◽

H Du ◽

H L Ozer

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Chinese Hamster ◽

Human Adenovirus ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Dependent Manner ◽

Viral Dna ◽

Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

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Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽

10.1093/genetics/148.3.989 ◽

1998 ◽

Vol 148 (3) ◽

pp. 989-1005 ◽

Cited By ~ 5

Author(s):

Keiko Umezu ◽

Neal Sugawara ◽

Clark Chen ◽

James E Haber ◽

Richard D Kolodner

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Protein A ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Physical Analysis ◽

Single Stranded Dna ◽

Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

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Mutational blockage of DNA synthesis in Paramecium tetraurelia

Canadian Journal of Zoology ◽

10.1139/z76-242 ◽

1976 ◽

Vol 54 (12) ◽

pp. 2089-2097 ◽

Cited By ~ 19

Author(s):

E. L. Peterson ◽

J. D. Berger

Keyword(s):

Dna Synthesis ◽

Deoxyribonucleic Acid ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Paramecium Tetraurelia ◽

Selection System ◽

Replication Process ◽

Temperature Sensitive Mutants ◽

Fold Enrichment ◽

Comparison Of Experiments

One hundred and ninety-eight temperature-sensitive mutants of Paramecium tetraurelia were isolated after nitrosoguanidine mutagenesis. In some experiments, mutants were recovered with the aid of a bromouracil (BU) selection system. Fifty-six mutants showed cessation of cell division within one cell cycle at the restrictive temperature and were designated ts-0. Fourteen of the ts-0's showed a greater than 90% reduction in rnacronuclear deoxyribonucleic acid (DNA) synthesis at the restrictive temperature. Two ts-0. DNA-defective lines continued protein synthesis at greater than 50% the normal rate after arrest of DNA synthesis. Hence, these two mutants may be directly affected in the replication process itself. The two mutants are allelic and, in addition, a third 'leaky' allele was recovered. Comparison of experiments in which either BU selection or no selection was employed shows that a greater than 10-fold enrichment for ts mutants resulted from BU selection.

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RCC1, a regulator of mitosis, is essential for DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3337 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3337-3345 ◽

Cited By ~ 50

Author(s):

M Dasso ◽

H Nishitani ◽

S Kornbluth ◽

T Nishimoto ◽

J W Newport

Keyword(s):

Dna Replication ◽

Regulatory System ◽

Sperm Chromatin ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Replication Complex ◽

Temperature Sensitive Mutants ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.

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MEIOTIC RECOMBINATION AND DNA SYNTHESIS IN A NEW CELL CYCLE MUTANT OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/90.1.49 ◽

1978 ◽

Vol 90 (1) ◽

pp. 49-68

Author(s):

Yona Kassir ◽

Giora Simchen

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Dna Synthesis ◽

Nuclear Division ◽

Temperature Shift ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Essential Function ◽

Normal Meiosis

ABSTRACT Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

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ISOLATION AND CHARACTERIZATION OF dnaX AND dnaY TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI

Genetics ◽

10.1093/genetics/92.4.1041 ◽

1979 ◽

Vol 92 (4) ◽

pp. 1041-1059

Author(s):

Joan M Henson ◽

Herman Chu ◽

Carleen A Irwin ◽

James R Walker

Keyword(s):

Escherichia Coli ◽

Dna Synthesis ◽

Fold Increase ◽

Temperature Sensitive ◽

E Coli ◽

Temperature Sensitive Mutants ◽

Isolation And Characterization ◽

Ts Mutations

ABSTRACT Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX + and Y +; the shorter F210 carries dnaY +, but not X +. Lambda transducing phages that carry dnaX + or Y + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

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THE CHARACTERISTICS AND GENETIC MAP LOCATION OF A TEMPERATURE SENSITIVE DNA MUTANT OF E. COLI K12

Genetics ◽

10.1093/genetics/72.4.569 ◽

1972 ◽

Vol 72 (4) ◽

pp. 569-593

Author(s):

Beverly Wolf

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Chain Elongation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Bacterial Dna ◽

E Coli ◽

Dna Chain ◽

Inhibition Of Dna Synthesis ◽

Dna Mutant

ABSTRACT A temperature sensitive strain of E. coli K12 has been isolated in which residual DNA synthesis occurs at the 40°C restrictive temperature; syntheses of RNA, protein and DNA precursors are not directly affected. The mutation has been designated dna-325 and is located at 89 min on the E. coli map in the same region where the dnaC locus is found. dnaC mutants are considered to be defective in DNA initiation. Some of the data are consistent with the view that the dna-325 mutation is temperature sensitive in the process of DNA initiation rather than DNA chain elongation: (1) more than two cell divisions occur after a shift to 40°C; (2) upon a shift down to 30°C, cell division occurs again only after the DNA content of the cells has doubled; (3) 80% more DNA is made at 30°C in the presence of chloramphenicol after prior inhibition of DNA synthesis at 40°C. These three observations indicate that rounds of DNA replication were completed at 40°C. Also (4) infective λ particles can be made at 40°C long after bacterial DNA replication has ceased. It appears however that some DNA initiation can occur at 40°C since (1) a limited amount of DNA synthesis does occur at 40°C after prior alignment of the chromosomes by amino acid starvation at 30°C, and (2) after incubation in bromouracil at the restrictive temperature, heavy DNA is found with both strands containing bromouracil.

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Mitotic mutants ofAspergillus nidulans

Genetics Research ◽

10.1017/s0016672300016049 ◽

1975 ◽

Vol 26 (3) ◽

pp. 237-254 ◽

Cited By ~ 181

Author(s):

N. Ronald Morris

Keyword(s):

Aspergillus Nidulans ◽

Nuclear Division ◽

Complementation Group ◽

Gene Symbol ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Temperature Sensitive Mutants ◽

Ofaspergillus Nidulans ◽

Complementation Groups ◽

Genetic Clustering

SUMMARYForty-five temperature-sensitive mutants ofAspergillus nidulanswhich are defective in nuclear division, septation or distribution of nuclei along the mycelium have been isolated, and most have been subjected to complementation analysis and mapped to chromosome. Thirty-five of the mutants were unable to complete nuclear division at the restrictive temperature. Twenty-six of these mutants exhibited a co-ordinate drop in both spindle and chromosome mitotic indices at 42 °C, indicating that they fail to enter mitosis. These mutants have been assigned to the gene symbolnim. Nine mutants exhibited a co-ordinate rise in spindle and chromosome mitotic indices at 42 °C, indicating that they are arrested in mitosis. These mutants were assigned the gene symbolbim. Five mutants failed to form septa and were given the gene symbolsep; and five mutants had an abnormal nuclear distribution and were given the gene symbolnud. All of the mutations were recessive. Most of the mutants were in different complementation groups. Mutants in the same complementation groups were phenotypically similar, but phenotypically similar mutants were not necessarily or usually in the same complementation group. There was no evidence for genetic clustering of phenotypically similar mutants. The mutants were located on all eight chromosomes.

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