Properties of an in vitro system for studying temperature-sensitive DNA synthesis in ts A1S9 mouse L-cells

Abstract

The in vitro DNA synthesis has been observed in whole cell lysates and in cytosol and nuclear fractions of wild-type (WT-4) mouse L-cells and ts A1S9 cells which exhibit temperature-sensitive (ts) DNA replication in vivo. The product, labelled with substrate 3H-labelled TTP, is resistant to alkali and has the buoyant density (1.709 g/cm3) expected for normal mouse DNA. Pulse-chase studies, in which newly made, single-stranded DNA was analyzed by velocity sedimentation in alkaline sucrose density gradients, revealed that in vitro DNA synthesis proceeds by a discontinuous mechanism. Approximately half of the DNA made in a 30-s pulse sedimented at 3–8S; the rest was very heterogeneous with S values between [Formula: see text] and 30S. After incubation for up to 300 s, a majority of the newly made DNA (>85%) sedimented as the larger, heterogeneous material, with some cosedimenting with chromosomal size DNA.The ts DNA synthesis phenotype of ts A1S9 cells is expressed in vitro. Thus, the activity of extracts of ts cells incubated at the nonpermissive (38.5 °C) temperature was commensurate with the in vivo activity. Restriction of the ts phenotype to DNA synthesis is evident in vitro since the RNA synthetic activity of lysates of temperature-inactivated ts A1S9 cells was equivalent to that of extracts obtained from cells grown at the permissive temperature (33.5 °C). The DNA synthetic activity of nuclei from WT-4 or ts A1S9 cells grown at 33.5 °C plus homologous cytosol is equivalent to that of the whole lysate. In contrast, such cytosol preparations give little, if any, enhancement of the activity of nuclei from ts A1S9 cells incubated at 38.5 °C for 16 h. The cytosol of such temperature-inactivated cells, which are almost fully effective with nuclei of control cells, produce little or no enhancement of DNA synthesis by homologous nuclei.