In vitro HeLa cell DNA synthesis similarity to in vivo replication

Fraser, J.; Huberman, Joel A.

doi:10.1016/0022-2836(77)90033-x

Cited by 38 publications

(9 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, the actual rate is probably closer to 50% of the in vivo rate . This is similar to the estimate of Fraser and Huberman (7) obtained by fiber autoradiographic analysis in a lysed cell system .…”

supporting

confidence: 89%

Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.

Hand¹,

Gautschi²

1979

The Journal of Cell Biology

View full text Add to dashboard Cite

We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system . CHO cells were treated with 0 .01% Brij-58 and the lysates were incubated at 30°C in a complete reaction mixture for in vitro DNA synthesis with [ 3H)thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 Jim, very similar to the size of units found in vivo . The rate of replication fork movement was 25-50% of the in vivo rate . More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min . This suggests that synthesis is halted by premature terminations . Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system . KEY WORDS in vitro DNA synthesis " DNA fiber autoradiography " in vitro initiation replication unit size replication fork movement rateIn vitro synthesizing systems have been of immense value in the study of DNA replication in prokaryotic cells (25) and papovavirus-infected mammalian cells (21). Mammalian cellular DNA replication has also been studied in vitro but not to the same extent . A variety of systems have been developed in which exogenous deoxynucleoside triphosphates are used as the direct precursors of cellular DNA; these include permeabilized cells (1,2,5,20), lysed cells (7, 19), isolated nuclei (14, 18, 23), and chromatin (17, 22) . In most of these, DNA synthesis has been shown to be semiconservative and a continuation of the in vivo process . J . CELL BIOLOGY

show abstract

supporting

confidence: 89%

Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.

Hand¹,

Gautschi²

1979

The Journal of Cell Biology

View full text Add to dashboard Cite

show abstract

“…ATP or GTP is used to initiate RNA primer synthesis at a frequency that depends on their relative concentrations (281) and is proportional to the rate of concurrent DNA synthesis (69). However, addition of rNTPs to nuclei never stimulated DNA synthesis more than 20-30% (69,167,169,213) unless nuclei were washed in 0.27 M NaCI (295), which resulted in a two to threefold stimulation. This can be explained by the ability of dNTPs to substitute for rNTPs at low rNTP concentrations (69), and by a failure to eliminate endogenous sources of rNTPs.…”

Section: Rna Primersmentioning

confidence: 99%

Replication of Eukaryotic Chromosomes: A Close-up of the Replication Fork

DePamphilis¹,

Wassarman²

1980

Annu. Rev. Biochem.

318

106

View full text Add to dashboard Cite

“…The procedure was adapted from Fraser and Huberman (1977). Lysates prepared from three dishes stimulated by PGFz, and insulin were pooled and prewarmed to 37°C.…”

Section: Alkaline Sucrose Gradientsmentioning

confidence: 99%

Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: Comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase α activity

Otto

Smith

Asúa

1988

Journal Cellular Physiology

View full text Add to dashboard Cite

An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2 alpha, insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca++-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37 degrees C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca++. Using various templates, it was shown that the increase in activity of DNA polymerase alpha correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase beta activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.

show abstract

In vitro HeLa cell DNA synthesis similarity to in vivo replication

Cited by 38 publications

References 41 publications

Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.

Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.

Replication of Eukaryotic Chromosomes: A Close-up of the Replication Fork

Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: Comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase α activity

Contact Info

Product

Resources

About