In vitro HeLa cell DNA synthesis II. Partial characterization of soluble factors stimulating nuclear DNA synthesis

Abstract

When HeLa cells are lysed in hypotonic buffer and the lysate is fractionated by centrifugation into particulate and soluble components, the particulate component (nuclei) is deficient compared to unfractionated lysate in the extent to which it can carry out in vitro DNA synthesis. In addition, rapidly labeled short DNA chains (Okazaki pieces) accumulate in purified nuclei, and are chased into higher molecular weight DNA to a lesser degree than in unfractinated lysate. When purified nuclei were reconstituted with soluble component, the capacity of the nuclei for in vitro DNA synthesis was fully restored, as was the capacity of the nuclei for conversion of Okazaki pieces to higher molecular weight DNA. This suggests that the soluble component contains a factor or factors necessary for normal DNA replication. The major incorporation-stimulating activity was partially characterized and partially purified from the soluble component. It is heat labile, non-dialyzable, partially recoverable in the supernatant after pH 5 precipitation, found mainly in a 55–85% saturated (NH 4) 2SO 4 fraction, and is included on Sephadex G-100. After passage through Sephadex G-100, the activity displays increased instability to storage at either 4°C or −70°C. Part of the activity does not bind to phosphocellulose at pH 7.2 and low salt; no additional activity can be recovered in a 0.5 M KCl wash of the phosphocellulose column. The Okazaki-piece-joining activity was found, along with the bulk of the incorporation-stimulating activity, in the 55–85% (NH 4) 2SO 4 fraction. These findings provide some of the groundwork for future attempts to completely purify and characterize those activities in the soluble component of cell lysates which are involved in DNA replication.