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

Abstract

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

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

“…Furthermore, at the normal rate of polymerization (50 to 100 bases per s) (32), Okazaki units are finished within a few seconds. This period might be prolonged to around 30 s or 1 min if the polymerization rate in vitro is 10 times slower than that in vivo (14,22,35). As the time course of the cell-free synthesis shows a linear increase during the first 15 min (Fig.…”

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

“…The in vitro replication system used is well characterized and active for a number of rodent cell lines (2,3,(10)(11)(12). It continues semiconservative DNA synthesis at an initial rate of up to 50% of that in intact cells (10) and probably supports some initiation as well (2, 4).…”

Correction of the Defect in Initiation of DNA Replication in a Temperature-Sensitive Mutant Hamster Cell Line by In Vitro Addition of Extracts from Normal Cells

“…We have found that the ts BN-2 phenotype is expressed in vitro (submitted for publication). The in vitro replication system used is well characterized and active for a number of rodent cell lines (2,3,(10)(11)(12). It continues semiconservative DNA synthesis at an initial rate of up to 50% of that in intact cells (10) and probably supports some initiation as well (2,4).…”