DNA synthesis in isolated HeLa cell nuclei. Evidence for in vitro initiation of synthesis of small pieces of DNA and their subsequent ligation

Abstract

Optimum conditions for a DNA synthesizing system based on isolated nuclei have been described (Krokan, H., Bjorklid, E., and Prydz, H. (1975), Biochemistry, preceding paper in this issue) [3H]TTP-labeled nascent DNA produced during very short pulses was analyzed by centrifugation in alkaline sucrose gradients. More than 80% of the radioactivity appeared in 2-4S pieces (primary DNA pieces). It would therefore seem that the synthesis of DNA is discontinuous both in the 5' leads to 3' and in the 3' leads to 5' directions. The size of the primary DNA pieces increases from 2-4 S up to 14 S with increasing pulse length. Evidence is presented that this increase is not caused by ligation between 2-4S primary pieces. Pulse-chase experiments showed that in this nuclear system primary pieces were ligated to a product generally larger than 30 S. Evidence is also given for the initiation of primary DNA pieces in vitro.