Assembly of secondary intermediates during deoxyribonucleic acid replication in transformed human fibroblasts

The elongation of replicative DNA was studied in transformed WI-38 cells (designated 2RA). Shear effects were avoided by use of an alkaline sucrose gradient sedimentation method whereby cells were lysed directly on top of gradients, at 4 degrees C in the dark. The earliest detected intermediate is a short (2 S) piece of DNA which is converted first to a 25 S piece and then to a 100 S piece, within 10 min. The 100 S piece is next converted to a 212 S, and a 370 S, and finally to a chromosomal DNA of about 450 S. This pattern is quite different from that previously reported by us for normal WI-38 cells, where there was a 50 S intermediate which was not quickly converted into a much larger size, but which gradually elongated, by addition of smaller pieces, to a larger size, of 100 S.; another difference was the time required for formation of the 100 S piece, i.e., 75 min (Rawles, J.W., Jr. and Collins, J.M. (1977) J. Biol. Chem. 252, 4762-4766).