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

Abstract

Fiber autoradiography was used to examine DNA made in vitro in a lysed cell system. CHO [Chinese hamster ovary] cells were treated with 0.01% Brij-58 [polyoxyethylene ether of fatty alcohol] and the lysates were incubated at 30.degree. C in a complete reaction mixture for in vitro DNA synthesis with [3H]TTP. Fiber autoradiograms showed the DNA was synthesized on tandemly arranged replication units of average size of 20 .mu.m, very similar to the size of in vivo units. 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. Synthesis may be halted by premature terminations. New initiations were demonstrated by replication units with labeled origins in DNA synthesized in vitro in which radioactivity was omitted for the first 10 min of incubation. This, plus observations that the distance between initiation points (replication units 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.