A nuclear system from synchronized HeLa cells is described which continues DNA replication a t sites which were active in vivo. Under the defined optimal conditions the initial rates of DNA replication in isolatednuclei range from 50 to lOOO/, of that in the cells of origin; 1-Z0/, of the HeLa DNA can be replicated in vitro. Proteins of the cytosol are essential to the stabilization of the replication system. Experiments in which the parental strands were fractionally labelled with bromodeoxyuridine provide evidence that the ordered nature of DNA replication in vivo is preserved in the system in vitro. Repair replication of homopolymer synthesis was insignificant under the conditions of study.Previous studies from this laboratory have described a nuclear system from synchronized HeLa cells which exhibits an S-phase specific incorporation of Requirements for the four deoxynucleotide triphosphates (dTTP, dGTP, dATP and dCTP), ATP, Mg2+ and some soluble protein factors from the cytoplasm have been documented as well as a striking dependency on ATP; the latter requirement is shared with the subcellular systems from procaryotes [5-81. Under optimum conditions the nuclear system engages in DNA synthesis a t 50 to 100°/, of the rate in vivo for the cells of origin and l-Zo/, of the HeLa genome can be replicated in vitro. Studies with nuclei from S-phase cells which were synthesizing DNA in vivo with bromodeoxyuridine a t the time of isolation have revealed that the [3H]dTTP incorporation by the isolated nuclei is largely a continuation of DNA replication a t sites which were active in vivo. Repair synthesis was excluded as a significant factor in these results. Using nuclei from cells labelled in a previous S-phase so as to contain bromodeoxyuridine in the template strands of late-replicating DNA, it was shown that the ordered nature of replication in vivo was preserved during DNA replication in the isolated nuclei.These studies illustrate that the isolated HeLa nuclei provide a system of intermediate complexity
25'which can be used to assess the role of chromatin structure, nuclear membranes and cytoplasmic factors in DNA replication in eucaryotes.
MATERIALS AND METHODS
Cell Culture and SynchronizationGrowth and maintenance of HeLa cells in shaker culture have been previously described [S]. Cells were synchronized by the addition of 1 pM amethopterin and 50 pM adenosine. The cultures were reversed by the addition of thymidine (1 mg/l) or bromodeoxyuridine (1.2 mg/l).Isolation and Assay of the Nuclei 2 h after reversal, unless otherwise stated, the cells were harvested by centrifugation a t 800 x g for 5 min a t 2-4 "C (hereafter 800 x g will refer to these conditions). The cells were washed in buffer A (10 mM Tris pH 7.8, 1 mM EDTA, 4 mM MgC1, and 6 mM 2-mercaptoethanol) a t 2 x lo6 cells/ml.The cells were then suspended in buffer A a t 70 x lo6 cells/ml. After swelling in the hypotonic buffer A for 10 min at 0 "C, the cells by were lysed 10-15 strokes in a Dounce homogenizer (Kontes Glass Corp.) and observed under a ...
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