scholarly journals Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

1995 ◽  
Vol 15 (1) ◽  
pp. 165-172 ◽  
Author(s):  
M Seki ◽  
T Kohda ◽  
T Yano ◽  
S Tada ◽  
J Yanagisawa ◽  
...  
Keyword(s):  
Dna Replication ◽  
Atpase Activity ◽  
Dna Synthesis ◽  
Dna Helicase ◽  
Chain Elongation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Dependent Atpase ◽  
Mutant Cells

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly

1991 ◽  
Vol 11 (9) ◽  
pp. 4669-4678 ◽  
Author(s):  
P A Kolodziej ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Mutant Enzyme ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Gradient Fractionation ◽  
Mutant Cells

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

scholarly journals Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

1991 ◽  
Vol 11 (9) ◽  
pp. 4669-4678 ◽  
Author(s):  
P A Kolodziej ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Mutant Enzyme ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Gradient Fractionation ◽  
Mutant Cells

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

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

Genetics ◽  
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.

scholarly journals The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

2007 ◽  
Vol 189 (24) ◽  
pp. 8793-8800 ◽  
Author(s):  
Christopher J. Rosario ◽  
Mitchell Singer
Keyword(s):  
Dna Replication ◽  
Developmental Process ◽  
Myxococcus Xanthus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Temperature Growth ◽  
Developmental Program

ABSTRACT Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaBA116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaBA116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaBA116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

scholarly journals Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

1984 ◽  
Vol 4 (9) ◽  
pp. 1815-1822 ◽  
Author(s):  
G C Zeng ◽  
J Donegan ◽  
H L Ozer ◽  
R Hand
Keyword(s):  
Dna Synthesis ◽  
Mutant Cell ◽  
Chain Elongation ◽  
Initial Increase ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
3T3 Cells ◽  
Mutant Cells

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells

1984 ◽  
Vol 4 (9) ◽  
pp. 1815-1822
Author(s):  
G C Zeng ◽  
J Donegan ◽  
H L Ozer ◽  
R Hand
Keyword(s):  
Dna Synthesis ◽  
Mutant Cell ◽  
Chain Elongation ◽  
Initial Increase ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
3T3 Cells ◽  
Mutant Cells

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.

scholarly journals TEMPERATURE-SENSITIVE MUTANTS OF A CHINESE HAMSTER CELL LINE. I. SELECTION OF CLONES WITH DEFECTIVE MACROMOLECULAR BIOSYNTHESIS

Genetics ◽  
1975 ◽  
Vol 80 (3) ◽  
pp. 549-566
Author(s):  
Donald J Roufa ◽  
Susan J Reed
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Protein Biosynthesis ◽  
Chinese Hamster ◽  
Selection Procedure ◽  
Brdu Incorporation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature

ABSTRACT Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxy-uridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39° and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39° to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115-47 and 115-53) also stop incorporating radioactive amino acids and uridine within six hours at 39°. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33° after 8 hours at 39°, both resumed DNA synthesis immediately (< 1 hour). Reversal of defective DNA synthesis in both mutants was sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication.

scholarly journals Targeting the DNA replication stress phenotype of KRAS mutant cancer cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tara Al Zubaidi ◽  
O. H. Fiete Gehrisch ◽  
Marie-Michelle Genois ◽  
Qi Liu ◽  
Shan Lu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Stress ◽  
Proton Radiation ◽  
Wild Type ◽  
Cellular Effects ◽  
Proton Treatment ◽  
Dna Replication Stress ◽  
Fork Stalling ◽  
Mutant Cells

AbstractMutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.

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