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Hand R, Gautschi JR. Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography. J Cell Biol. 1979;82(2):485-93doi: 10.1083/jcb.82.2.485.
Hand, R., & Gautschi, J. R. (1979). Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography. The Journal of cell biology, 82(2), 485-93. https://doi.org/10.1083/jcb.82.2.485
Hand, R, and Gautschi, J R. "Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography." The Journal of cell biology vol. 82,2 (1979): 485-93. doi: https://doi.org/10.1083/jcb.82.2.485
Hand R, Gautschi JR. Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography. J Cell Biol. 1979 Aug;82(2):485-93. doi: 10.1083/jcb.82.2.485. PMID: 479312; PMCID: PMC2110453.
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J Cell Biol. 1979 Aug;82(2):485-93. doi: 10.1083/jcb.82.2.485.

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

The Journal of cell biology

R Hand, J R Gautschi

PMID: 479312 PMCID: PMC2110453 DOI: 10.1083/jcb.82.2.485
Free PMC Article

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 degrees 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 micrometers, 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.

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