Semiconservative DNA replication in vitro. II. Replicative intermediates of mouse P-815 cells

Abstract

DNA chain growing during semiconservative replication was studied using both in vitro systems described in the preceding paper (preceding paper, ref 1) 3H-Labeled, 4-S Okazaki fragments synthesized in vivo just prior to permeabilization or lysis with Brij-58 were metabolically stable and quantitatively chased into high molecular weight DNA (20--100 S) during a subsequent incubation in vitro. Thus, DNA replication continued in vitro at the same growing points that were active in vivo. After a 20-s pulse at 30 degress C in vitro, more than 50% of incorporated radioactivity was found in the 4 S region of alkaline sucrose gradients suggesting a totally discontinuous mode of DNA chain growth. If the pulse were followed by a 1-min chase, 4-S molecules were converted into 6--12-S intermediates which upon continued incubation were joined with growing 20--100-S molecules (replicon-sized chains). Formation of all three classes of replicative intermediates, Okazaki fragments, 6--12-S intermediates, and 20--100-S molecules, occurred in vitro at least during the first 20 min. During this time, average rates of DNA chain growth and overall DNA synthesis were reduced to about the same extent, if compared to rates of intact cells. Thus, reduced chain growth rates appear to reflect primary deficiences of our in vitro systems, while initiation of replicative intermediates still occurs.