DNA synthesis in Balb/C-3T3 ts 2 cells is restricted by a temperature-sensitive function of late G1 phase

Biochemistry ◽  
1986 ◽  
Vol 25 (6) ◽  
pp. 1208-1216 ◽  
Author(s):  
Rose Sheinin ◽  
Dennis Mirjah ◽  
Margaret Dubsky ◽  
Jose Sigouin
Keyword(s):  
Dna Synthesis ◽  
G1 Phase ◽  
Temperature Sensitive ◽  
Sensitive Function

Induction of G1- cells at high frequency

1979 ◽  
Vol 40 (1) ◽  
pp. 33-42
Author(s):  
P.M. Naha
Keyword(s):  
Dna Synthesis ◽  
Ultraviolet Irradiation ◽  
High Frequency ◽  
Generation Time ◽  
G1 Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Experimental Conditions ◽  
G1 Cells

Experimental conditions are reported on the induction of G1- cells at high frequency by ultraviolet irradiation of temperature-sensitive cells arrested in G1 phase at the restricted temperature. Neither the wild type cells of Balb/C-3T3 nor the temperature-sensitive derivative of tsA83 grown at the permissive temperature could be ‘mutated’ under similar conditions. One such ‘mutant’ (RI8) was ‘inducible’ from a G1+ phenotype at 33 degrees C to G1- state at 38 degrees C within one cell generation time. The ‘inducible’ property of RI8 from G1+ to G1- lends support to the theory that the duration of the G1+ phenotype is determined by the level of precursors for DNA synthesis. Since the G1- variants were isolated from revertants, the frequency of ‘mutagenesis’ appeared to be artifically high.

scholarly journals Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
Author(s):  
Xuan Wang ◽  
Grzegorz Ira ◽  
José Antonio Tercero ◽  
Allyson M. Holmes ◽  
John F. X. Diffley ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Dna Ligases ◽  
Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

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

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.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity

1990 ◽  
Vol 10 (11) ◽  
pp. 5688-5699
Author(s):  
B E Wojcik ◽  
J J Dermody ◽  
H L Ozer ◽  
B Mun ◽  
C K Mathews
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster Ovary ◽  
Reductase Activity ◽  
Chinese Hamster ◽  
Temperature Shift ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Dntp Pool ◽  
Ribonucleotide Reductase Activity

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

Sign in / Sign up

Export Citation Format

Share Document