Nuclear protein from cultured hepatoma cells preferentially inhibits copying of denatured DNA by isolated chromatin

Abstract

productionDNA synthesis is conducted in vitro by isolated chromatin from a variety of eukaryotic cells [l--5]. Unlike intact cells or isolated nuclei, the chromatin system is free of permeability barriers which restrict passage of macromolecules to the site of DNA replication. We have thus been able to show that the chromatin-associated replicative system copies exogenous DNA templates at a high efficiency [S] . Of particular interest is the observation that linear DNA, which serves as a poor template for solub~~ed DNA polymerases is replicated at a high rate by chromatin-associated DNA polymerase (Y [5]. We describe here a nuclear protein, isolated from cultured rat hepatoma cells which preferentially inhibits copying of single-stranded DNA by chromatin. Materials and methods CellsRandomly growing rat hepatoma cultured (HTC) cells were cultivated in suspension in Swim's S-77 medium which contained 10% new-born calf serum [6]. Preparation of chromatin and of soluble nuclear proteinsChromatin was isolated from washed nuclei [7], as in [4]. The ratio of DNA to protein in the various chromatin preparations was 0.07-0.1. Crude extracts of nuclear proteins were prepared as in [8]. These extracts served as source for soluble DNA polymerase activity which was 90-95%inactivated in the presence of 2 mM Nethyl maleimide, thus represent~g mostly DNA polymerase ar and perhaps r polymerase activity [9]. A protein which inhibits copying of exogenous DNA templates by chromatin-associated polymerases was partially purified and freed of DNA polymerases by heating the crude nuclear extract at 75°C for 10 mm. Denatured proteins were removed by centrifugation at 90 000 X g for 30 min and the clear supernatant served as source of inhibitory activity. When stored at -6O"C, the inhibitor remained active for at least 2 months. Other methodsAmount of protein was dete~ned by the Lowry method [lo]. ATP concentration was estimated by the lucifer~-luciferase assay adapted to liquid scintillation spectrometer [ 111. Chromatin DNA was determined as in [ 121. The heat-treated nuclear extract was filtered on 1.5 X 70 cm column of Sephadex G-200 as in [8]. Results and discussionCrude extract of nuclei of HTC cells contain activity which suppresses the in vitro copying of 265

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase .beta.

“…Chromatin-associated DNA polymerases are able to copy defined exogenous DNA templates at high efficiency (Kaftory & Fry, 1978). This copying of exogenous templates by chromatin requires ATP; its rate of chain elongation is related to the in vivo rate of DNA replication, and the rate of the reaction can be specifically modulated by nuclear proteins (Kaftory & Fry, 1978;Kaftory et al, 1979; Weisman-Shomer et al, 1979). Thus, certain properties of the in vivo replicative machinery are better reflected by chromatin than by isolated DNA polymerases.…”

“…Particularly with respect to physiological problems, it seems important to measure accuracy in crude preparations containing DNA polymerases which may better reflect the in vivo fidelity of DNA synthesis. An appropriate system for such measurements are chromatin-associated polymerases, which are capable of copying defined exogenous DNA templates (Kaftory & Fry, 1978;Kaftory et al, 1979;Weisman-Shomer et al, 1979). A major obstacle in assessing the fidelity of replication of defined synthetic polynucleotides by polymerases conjoined with chromatin is the presence of large amounts of genomic DNA within the chromatin.…”