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Studies on in Vitro DNA Synthesis.* Purification of the Dna G Gene

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Proc. Nat. Acad. Sci. USA Vol. 70, No. 5, pp. 1613-1618, May 1973 Studies on In Vitro DNA Synthesis.* Purification of the dna G Gene Product from Escherichia coli (dna A, dna B, dna C, dna D, and dna E gene products/+X174/DNA replication/DNA polymerase III) SUE WICKNER, MICHEL WRIGHT, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by Alfred Gilman, March 12, 1973 ABSTRACT q5X174 DNA-dependent dNMP incorpora- Hirota; BT1029, (polA1, thy, endo I, dna B ts) and BT1040 tion is temperature-sensitive (ts) in extracts of uninfected endo I, thy, dna E ts), isolated by F. Bonhoeffer and E. coli dna A, B, C, D, E, and G ts strains. DNA synthesis (polAi, can be restored in heat-inactivated extracts of various dna co-workers and obtained from J. Wechsler; PC22 (polA1, his, ts mutants by addition of extracts of wild-type or other strr, arg, mtl, dna C2 ts) and PC79 (polAi, his, star, mtl, dna D7 dna ts mutants. A protein that restores activity to heat- ts), derivatives (4) of strains isolated by P. L. Carl (3) and inactivated extracts of dna G ts cells has been extensively obtained from M. Gefter. DNA was prepared by the purified. This protein has also been purified from dna G ts OX174 cells and is thermolabile when compared to the wild-type method of Sinsheimer (15) or Franke and Ray (16). protein. The purified dna G protein has a molecular weight of about 60,000, is insensitive to N-ethylmaleimide, and Preparation of Receptor Crude Extracts. Cells were grown to binds poorly to DNA. It does not stimulate heat-inacti- an OD595 of 0.45 at 300 in Hershey broth plus thymine (10 vated crude extracts of dna B, C, D, or E ts cells and lacks pig/ml) and thiamine (10,ug/ml), collected by centrifugation at detectable RNA and DNA polymerase activities. room temperature (250), resuspended in 0.002 volume of 0.05 Mutants of Escherichia coli have been isolated that are tem- M Tris HCl (pH 7.5) and 10% sucrose, and frozen in a dry perature-sensitive for DNA replication, and the genes in- ice-ethanol bath (12). Frozen cells were thawed in an ice- volved have been designated dna A, B, C, D, E, F, and G (1-3) water bath, incubated with 0.2 mg/ml lysozyme and 0.2% The product of the dna E gene is DNA polymerase III (4, 5) Brij 58 for 30 min at 00, and centrifuged for 30 mMi at 50,000 while that of the dna F gene is ribonucleotide reductase (6). X g at 4°. After centrifugation, the supernatant was frozen In vitro DNA-synthesizing systems have been developed that in small portions, and is designated receptor crude extracts. depend on one or more of the products of these dna genes (7- Complementation Assay. Each assay (0.075 ml) contained 12). One of these systems, in which crude extracts of E. coli 20 mM Tris* HCl (pH 7.6), 10 mM MgCl2, 4 mM dithiothreitol catalyze the conversion of OX174 single-stranded circular DNA to the double-stranded replicative form depends on the products of dna A, B, C, D, E, and G genes (11, 12). This has TABLE 1. Purification dna been shown by the increased thermolability of OX174 DNA- of G gene product dependent dNMP incorporation in extracts from ts cells when Total Specific % compared to extracts from temperature-resistant revertant protein Total activity Re- cells. The stimulation of inactive crude extracts of dna ts Fraction (mg) U (U/mg) covery cells by fractions from wild-type or the other ts cells has provided a complementation assay for the purification of the High-speed dna gene products. The purpose of this communication is to supernatant 59,500 - - - report the isolation of the dna G gene product of E. coli by this 20-30% Ammonium assay and its characterization. Using a different complementa- sulfate fraction 3,240 1375 0.37 100 tion assay employing E. coli concentrated on cellophane DNA-agarose discs, Ntisslein et al. purified an activity that stimulates DNA eluate 100 727 6.7 53 replication by dna G ts cells (13) and another activity that DEAE-cellulose stimulates DNA replication by dna E ts cells (5). eluate 28.8 650 22.6 47 DEAE-sephadex MATERIALS AND METHODS eluate 3.8 270 74 20 Bacterial Strains and DNA. The following E. coli strains Glycerol gradient* 1.3 270 222 20 were used: NY73 (polA1, thy, leus, metE, rifp, strr, dna G3 ts), a derivative (4) of PC3 (3) obtained from J. Wechsler; HMS- The results presented above were obtained with dna G receptor 83 (polAl, polBl, thy, lys) (14), obtained from C. C. Richard- crude extracts (E. coli strain NY73) which had been frozen and thawed to inactivate the thermolabile dna G gene product. son; CRT4638 (polAl, endo I, thy, dna A ts), obtained from Y. * This step was performed with only part of the DEAE- sephadex eluate. We calculated thevalues reported assuming that * This is paper III; papers I and II in this series are refs. 12 and the yield would be the same if the entire fraction were subjected 22. to the glycerol-gradient procedure. 1613 Downloaded by guest on September 29, 2021 1614 Biochemistry: Wickner et al. Proc. Nat. Acad. Sci. USA 70 (1978) 0.04 mM each of dATP, dGTP, dCTP, and a32P dTTP (300- 7.5), 1 mM dithiothreitol, 0.5 mM EDTA, and 20% glycerol 500 cpm/pmol), 1.5 mM ATP, 0.05 mM each of UTP, CTP, (buffer A) and dialyzed against buffer A for 6 hr with six and GTP, 10 sg/ml rifampicin, 2 mM spermidine-HCl, 500 2-liter changes of buffer. pmol qX DNA, 0.05 ml of ts receptor crude extract (15 mg/ml of protein) inactivated by freezing and thawing or by heating DNA-agarose column chromatography 30 min at 300, and protein fractions as indicated. After incuba- The dialyzed ammonium sulfate fraction was diluted to 250 tion at 300 for 20 min the reaction was stopped and acid- ml with buffer A minus glycerol and applied to a 6 X 40-cm precipitated as described (12). One unit (U) of activity in- column of denatured calf-thymus DNA-agarose (18). It was corporated 1 nmol of dTMP in 20 min at 30° under the condi- essential that the salt concentration was 5 mM or lower be- tions described above. Specific activity refers to units of fore the sample was applied to the column. The column was activity per mg of protein; protein was measured by the washed with 500 ml of buffer A containing 10% glycerol, and method of Bucher (17). the dna G activity was eluted with buffer A containing 1 M RESULTS NaCl. The 1 M NaCl eluate was adjusted to 50% saturation with solid ammonium sulfate (29.1 g/100 ml); the precipitate Purification of dna G gene product-crude extract was collected by centrifugation and dissolved in 14 ml of 0.05 All purification steps were performed at 4°. E. coli HMS-83 M Tris- HCl (pH 7.8), 1 mM dithiothreitol 0.5 mM EDTA, (400 g), suspended in 400 ml of 0.02 M potassium phosphate and 20% glycerol (buffer B). (pH 7.5), 0.05 M KCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 10% glycerol, was disrupted by passage through the DEAE-cellulose column chromatography Manton-Gaulin laboratory homogenizer at 9-10,000 lb/in2. The was 2 of The crude extract (900 ml) was centrifuged at 100,000 X DNA-agarose fraction dialyzed against liters g buffer B and applied to a DEAE-cellulose column (2.2 X 19 for 60 min and the pellet was discarded. cm) equilibrated with 2 liters of the same buffer. The column Streptomycin sulfate and ammonium sulfate was washed with 35 ml of buffer B and then developed with a precipitation 600-ml linear gradient from 0 to 0.35 M KCl in buffer B. A solution of 20% streptomycin sulfate was added to the The dna G activity eluted at 0.1 M KCl. crude extract to a final concentration of 4%, and the mixture was centrifuged at 10,000 X g for 15 min. The supernatant DEAE-sephadex column chromatography was adjusted to 40% saturation with solid ammonium sulfate The active fractions (36 ml) were pooled, dialyzed against 2 (22.6 g/100 ml); after centrifugation at 10,000 X g for 15 liters of buffer B, and applied to a DEAE-sephadex column min, the supernatant was removed and the pellet was washed (2 X 24 cm) equilibrated with the same buffer. The column successively with 200 ml each of 40%, 30%, and 20% was washed with 20 ml of buffer B and then developed with a saturated ammonium sulfate solution in 0.02 M potassium 400-ml linear gradient from 0 to 0.2 M KCl in buffer B. The phosphate (pH 7.5), 1 mM dithiothreitol, and 0.5 mM EDTA. dna G activity eluted between 0.17 and 0.19 M KCl; these The supernatant obtained after extraction with 20% ammo- fractions were pooled and adjusted to 50% saturation with nium sulfate was adjusted to 40% saturation with solid ammo- solid ammonium sulfate. The pellet obtained after centrifuga- nium sulfate (11.3 g/100 ml). The precipitate was collected by tion at 50,000 X g for 20 min, was dissolved in 3 ml of buffer centrifugation, dissolved in 100 ml of 0.02 M Tris HCl (pH B.
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