DNA synthesis in vitro in lysates of Escherichia coli

A system using crude lysates of Escherichia coli PolA- for in vitro synthesis of DNA has properties similar to those described previously for washed DNA - membrane complexes, i.e. short-lived synthesis of DNA, 2-3-fold stimulation by ATP, semiconservative form of synthesis, and most of the product in the size distribution of Okazaki fragments. The proximate substrate is shown to be the deoxynucleoside triphosphate. The lysate system can be fractionated into a particular membrane - DNA complex and a soluble portion, each of which is inactive by itself, but which upon mixing restores activity. The particulate fraction provides template as well as essential protein(s). The soluble fraction contains DNA polymerases II and III both of which are capable of the necessary polymerase function, and a factor that is required for the stimulation of DNA synthesis by ATP. The stimulatory activity can also be assayed by restoration of activity to an aged lysate, which has lost the ATP-dependent synthesis; the activity is not a kinase, and is not an ATP-dependent nuclease, at least of the type currently known. The ATP-dependent stimulatory factor has been partially purified but further purification or characterization of it has been limited by its extreme instability. Both the stimulatory factor and ATP are required for semiconservative synthesis in the lysate, and for synthesis of the short fragments. Similar to results in vivo and in permeable cells the new DNA appears to contain RNA, as judged by the evidence for RNA - DNA junctions from modified nearest neighbor experiments. However, the nucleotides at the RNA - DNA junctions in the lysate system are not specific, in contrast to results in permeabilized Escherichia coli.