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

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.

S 0 aE

cLCL a. I-

0.2 0.4 0.6 0.8 1.0 2.0 3.0 4.0 10 20 30 40 dna G 10,anrecentor t..Ucrude CAF'1%Fextrnet (ma) d G minutes -vw dnaG gene product (,O9) FIG. 1. Parameters of dTMP incorporation catalyzed by the dna G gene product. The assay conditions used were those described in Methods with dna G gene product purified through the DEAE-sephadex step. (A) The influence of dna G receptor crude extract. The amount of receptor crude extract was varied as indicated with 2.5 Mg, 0 O; 0.5 ,ug, A A; or 0 pg, *- of dna G gene product. (B) The influence of dna G gene product. The amount of dna G gene product was varied with 0.8 mg of receptor crude extract and either 500 pmol OX DNA, A A, or no ODNA, *-*. (C) The influence of time on the reaction. The time of incubation was varied using 0.6 sg of dna G gene product and 0.8 mg of dna G receptor crude extract with OX DNA plus dna G gene product, A A; with qX DNA without dna G gene product, * ; and without OX DNA with dna G gene product, 0 o.

Downloaded by guest on September 29, 2021 -'!-brsw Proc. Nat. Acad. Sci. USA 70 (1978) dna G Gene Product of Escherichia coli 1615

Glycerol-gradient centrifugation was nearly the same whether cells were lysed with Brij- A portion of the DEAE-sephadex fraction was dialyzed against lysozyme or by passage through the Manton-Gaulin homog- 0.05 M Tris- HCl (pH 7.8), 0.2 M KCl, 1 mM dithiothreitol, enizer. The extent of purification from the ammonium sulfate 0.5 mM EDTA, and 5% glycerol. The dialyzed sample (0.2 fraction through the glycerol-gradient step was about 600-fold ml) was layered on 6 ml of a 10-30% glycerol gradient in the with a 20% yield. same buffer and centrifuged in the Spinco SW 50.1 rotor at The stimulation of receptor crude extracts from dna A, B, 50,000 rpm for 35 hr. The dna G activity sedimented through C, D, E, and G ts cells by various fractions prepared during 2/3 of the gradient. the isolation of the dna G gene product was examined (Table The purification and yield of dna G activity carried through 2). The DNA-agarose step completely separated the dna B the above procedure are summarized in Table 1. Crude product from the other activities. The purification procedure extract prepared with the Manton-Gaulin homogenizer could resulted in a marked loss of dna C and D activities. The not be assayed directly because dTMP incorporation was in- DEAE-sephadex step removed dna E activity from dna G dependent of added OX DNA and receptor crude extract. gene product preparations. Some purified fractions (see below) However, when cells were lysed by the Brij-lysozyme method stimulated both dna G and dna A receptor crude extracts; in described above for the preparation of receptor crude extracts, these fractions both activities cosedimented in glycerol the activity was dependent on OX DNA and receptor crude gradients. As discussed further below this stimulation of dna A extract; close to 100% of the activity in such crude extracts receptor crude extract appears unrelated to the dna A gene could be recovered in the 20-30% ammonium sulfate fraction product. with a 20- to 50-fold purification. The recovery of activity in the 20-30% ammonium sulfate fraction per gram of cells Properties of the stimulation of dna G ts receptor extracts by dna G gene product The requirements for stimulation of dna G receptor crude ex- TABLE 2. Separation of dna A,B,C,D, and E gene products during the purification of dna G gene product tract by purified dna G gene product were examined (Table 3). The complete system required OX DNA and was dependent dna gene product measured on the amount of DNA added. With 0.015 U of dna G product dTMP (pmol) and 100, 500, or 2250 pmol of OX DNA, the system incor- Fraction from incorporated porated 4.1, 15.6, and 4.4 pmol of dTMP, respectively. High wild-type cells G A B C D E concentrations of ATP were also required for maximum 0-40% stimulation of receptor crude extracts. With 0.015 U of dna G ammonium activity and 0.3, 1.3, or 5.8 mM ATP, 5.6, 15.6, and 10.6 sulfate 18.2 17.8 33.7 16.8 19.6 17.0 pmol of dTMP were incorporated, respectively. The omission DNA-agarose of deoxynucleoside triphosphates had no effect on the reaction 0.01 M NaCl described in Table 3, presumably because they are present in eluate <0.5 <1.0 34.7 0.7 <0.1 0.4 the receptor crude extract. In contrast dNTPs were required DNA-agarose for XX DNA-dependent dNMP incorporation catalyzed by 1.0 M NaCl ammonium sulfate obtained from crude ex- eluate 20.4 20 <0. 5 3.0 0.3 32.1 dialyzed fractions DEAE-Sephadex 10.3 0.8 0.2 0.2 0.5 0.2 tracts (12). Spermidine has been reported to stimulate ->X DNA-dependent dNMP incorporation (11). In our hands, The ammonium sulfate fraction was prepared by lysing wild- this stimulation has been variable, and as shown in Table 3, type cells (E. coli strain HAIS-83) frozen in sucrose (as described spermidine stimulated dTMP incorporation about 2-fold. in Methods) with 0.2% Brij and 0.2 mg/ml lysozyme in an ice Under the conditions of the assay both purified dna G activity bath for 30 min, centrifuging at 100,000 X g for 30 min, and ad- and dna G receptor crude extract were limiting. As shown in justing the supernatant (20 ml) to 40% saturation with a neutral- Fig. 1A, with increasing amounts of receptor crude extract, ized saturated ammonium sulfate solution. After centrifugation, incorporation of dTMP increased in the presence of a constant the pellet was dissolved in 0.05 M Tris - HCl (pH 7.5), 1 mM di- amount of dna G activity. With a constant amount of dna G thiothreitol 0.a mM EDTA, and 10% glycerol (2 ml) and dialyzed 2 receptor crude extract, stimulation of receptor crude extract hr against this buffer. The DNA-agarose fractions and the DEAE- by the purified dna G gene product increased linearly with Sephadex fraction were prepared from HMS-83 cells as described for the purification of dna G gene product. 0.12 mg of ammonium increasing amount of dna G gene product to a plateau (Fig. sulfate fraction, 0.6 mg of DNA-agarose, 0.01 M NaCl eluate, 1B). The reaction was almost linear with time for at least 40 0.012 mg of DNA-agarose, 1.0 M NaCl eluate, and 0.14 lsg of min at 300 (Fig. 1C). The assay was salt-, glycerol-, and DEAE-sephadex fraction were assayed under the conditions sucrose-sensitive; 0.05 M KCl inhibited the reaction 50%; described in Methods with receptor crude extracts prepared from 10% glycerol (final) inhibited 50%; 7.5% sucrose (final) in- dna AB,C,D,E, or G cells. The ammonium sulfate fraction inhibited 50%. corporated no detectable dTMP in the absence of OX DNA and The acid-insoluble product (dTMP-labeled) formed during incorporated 0.9 pmol in the presence of DNA minus receptor the reaction was shown to be DNA by its stability to heating crude extract; the other fractions incorporated no detectable at 1000 for 10 min in 0.2 M NaOH as well as by its sensitivity dTMP in the presence or absence of OX DNA in the absence of to DNase. The product synthesized during the reaction sedi- receptor crude extract. Receptor crude extracts of dna A, B, C, D, mented in neutral sucrose gradients at the position of RF II or G ts cells incorporated 0.2-1 pmol of dTMP dependent onOX DNA in the absence of added fraction, while those of dna E ts and in alkaline sucrose gradients at the position of full-length cells incorporated 4.9 pmol. The values in the table have been linear strands (52%) as well as smaller pieces (48%). This size corrected for this endogenous activity of the receptor crude ex- distribution of the product was the same after 10, 20, or 40 tracts. min of incubation. Downloaded by guest on September 29, 2021 1618 Biochemistry: Wickner et al. Proc. Nat. Acad. Sci. USA 70 (1973)

The complementation assay, as described, above, -is also 7. Moses, R. & Richardson, C. C. (1970) Proc. Nat. Acad. Sci. Partially USA 67, 674-681. being used to purify the dna B gene product. 8. Schaller, H., Otto, B., Nusslein, V., Huf, J., Herrmann, R. & purified fractions, isolated from dna B ts cells, are thermolabile Bonhoeffer, F. (1972) J. Mol. Biol. 63, 183-200. compared to fractions isolated from dna C ts cells. Also by 9. Wickner, R. B. & Hurwitz, J. (1972) Biochem. Biophys. Res. this complementation assay, activities that stimulate dna C Commun. 47, 202-211. D crude extracts have been purified. Thus, 10. Wickner, W. T., Brutlag, D., Schekman, R. & Kornberg, A. and dna receptor (1972) Proc. Nat. Acad. Sci. USA 69, 965-969. by complementation in the OX174 DNA-dependent system, it 11. Schekman, R., Wickner, W. T., Westergaard, O., Brutlag, should be possible to purify the products of all the known dna D., Geider, K., Bertch, L. L. & Kornberg, A. (1972) Proc. genes. Because a defined DNA template is used, it should be Nat. Acad. Sci. USA 69, 2691-2695. possible to analyze the mechanism of DNA synthesis carried 12. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. (1972) Proc. Nat. Acad. Sci. USA 69, 3233-3237. out by this system. 13. Niisslein, V., Bonhoeffer, F., Klein, A. & Otto, B. (1972) in The Second Annual Harry Steenbock Symposium, eds. Wells, This work was supported by grants from the National Institute R. & Inman, R. (University Park Press, Maryland), in of Health (GM-13344) and the American Cancer Society (NP- press. 8900). S. W. is a Predoctoral trainee of The National Institute of 14. Campbell, J. L., Soll, L. & Richardson, C. C. (1972) Proc. Health, while M. W. is a Fellow of The Jane Coffin Childs Memo- Nat. Acad. Sci. USA 69, 2090-2094. rial Fund for Medical Research. 15. Sinsheimer, R. L. (1966) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper and 1. Hirota, Y., Ryter, A. & Jacob, F. (1968) Cold Spring Harbor Row, New York), pp. 569-576. Symp. Quant. Biol. 33, 677-693. 16. Franke, B. & Ray, D. S. (1970) Virology 44, 168-187. 2. Wechsler, J. A. & Gross, J. D. (1971) Mol. Gen. Genet. 113, 17. Bucher, T. (1947) Biochim. Biophys. Acta, 1, 292-314. 273-284. 18. Schaller, H., Niisslein, C., Bonhoeffer, F. J., Kurz, C. & 3. Carl, P. L. (1970) Mol. Gen. Genet. 109, 107-122. Neitzschmann, I. (1972) Eur. J. Biochem. 26, 474-481. 4. Gefter, M. L., Hirota, Y., Kornberg, T., Wechsler, J. & 19. Sigal, N., Delius, H., Kornberg, T., Gefter, M. L. & Alberts, Barnoux, C. (1971) Proc. Nat. Acad. Sci. USA 68, 3150- B. (1972) Proc. Nat. Acad. Sci. USA 69, 3537-3541. 3153. 20. Jones, 0. W. & Berg, P. (1966) J. Mol. Biol. 22, 199-209. 5. Niisslein, V., Otto, B., Bonhoeffer, F. & Schaller, H. (1971) 21. Wickner, R. B., Ginsberg, B., Berkower, I. & Hurwitz, J. Nature New Biol. 234, 285-286. (1972) J. Biol. Chem. 247, 489-497. 6. Fuchs, J. A., Karlstom, H. O., Warner, H. R. & Reichard, P. 22. Hurwitz, J., Wickner, S. & Wright, M. (1973) Biochem. (1972) Nature New Biol. 238, 69-71. Biophys. Res. Commun., 51, 257-267.

Correction. In the article "Structural and Functional Correction. In the article "Assembly of Rod-Shaped Virus Properties of Ribosomes Crosslinked with Dimethyl- In Vitro: Reconstitution with Cucumber Green Mottle suberimidate," by Slobin, L. I., which appeared in the Mosaic Virus Protein and Tobacco Mosaic Virus RNA," by Ohno, T., Inoue, H. & Okada, Y., which appeared in the December 1972 issue of Proc. Nat. Acad. Sci. USA 69, December 1972 issue of Proc. Nat. Acad. Sci. USA 69, 3769-3773, p. 3770, right-hand column, line 3 from top, 3680-3683, p. 3861, right-hand column, the first line should 10 mM KCl-6 mM 2-mercaptoethanol (pH 8.5) sh~uld read: "as 13 S Fig. 2B); whereas. . . "; the fifth line should read: 100 mM KCl-6 mM 2-mercaptoethanol (pH 8.5). read: "(Fig. 2A). The results. . . ". Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) dna G Gene Product of Esche*hia coli 1617

TABLE 3. Requirements for dna G compLmentation assay The purification procedures described (Table 1) resulted in the removal of all detectable ability to stimulate dna B, C, D, dTMP and E receptor crude extracts from preparations of purified incorporated dna G gene product. While a large amount of dna A comple- Additions (pmol) menting activity was removed during purification, dna G Complete 15.6 preparations retained the ability to stimulate dna A receptor Omit rifampicin 16.5 crude extracts. We believe that this activity is not due to the Omit spermidine 9.55 dna A gene product; purification of dna G activity from dna A Omit ATP 2.31 ts cells yielded dna A and G activities that showed no in- Omit dna G receptor crude <0.2 creased thermolability when compared with wild-type prepa- Omit 4,X174 DNA 0.47 rations. In contrast, purification of the dna G activity from Omit dna G gene product 1.2 dna G ts cells yielded dna A and G complementing activities that showed equally increased thermolability when compared The assay conditions were as described in Methods with 0.02 U with wild-type preparations. These results suggest some of dna G activity (DEAE-sephadex fraction). Reaction mixtures relationship between the dna G gene product and the ability were incubated for 30 min at 250. to activate dna A receptor crude extracts. Further evidence of the validity of the complementation the dna A gene product, since the activity when isolated from assay for the isolation of the dna gene products has been ob- dna A ts cells was not thermolabile. tained by a study of the relationship between dna E gene A comparison of thermolability was made between DEAE- product and DNA polymerase III. It has been demonstrated cellulose fractions purified from dna G ts cells (9.2 U/mg dna that DNA polymerase III is thermolabile when prepared G activity, 40% yield; 1.3 U/mg dna A activity, 4% yield) from dna E ts cells (4), that XX DNA-dependent dNMP and dna A ts cells (purity and yield as above) to determine if incorporation is thermolabile in crude extracts of dna E ts stimulation of the dna A receptor crude extract were due to cells (12), and that partially purified DNA polymerase III the dna G gene product. Both dna A and dna G complement- stimulates OX174 DNA-dependent dNMP incorporation by ing activities were thermolabile in the DEAE-cellulose frac- inactive crude extracts of dna E ts cells (11). We have purified tion from dna G ts cells (both were inactivated 50% after 3 the dna E activity through the DNA-agarose step, as de- min at 330) in comparison to that from dna A ts cells (both scribed above from dna E ts cells (BT1040) and wild-type cells were stable for more than 20 min at 33°). Thus, the purified (H560 F+). These two preparations and mixtures of them dna G gene product was able to stimulate dna A receptor were heated for various times at 370 and then assayed at 300 crude extracts. for dna E activity (under assay conditions described in Methods with dna E receptor crude extract) and for dNMP DISCUSSION incorporating activity (with DNase-treated salmon-sperm The requirement for dna A, B, C, D, E, and G gene products DNA template with and without 0.1 M KCl). The dna E in the conversion of single-stranded OX174 DNA to duplex complementing activity was inactivated 100% in the dna E DNA by extracts of E. coli has been used as an assay for ts preparation and 30% in the wild-type preparation, after purification of the dna G gene product. Specificity of comple- 15 min. The DNA polymerase activity was inactivated 90% mentation is not proof that a particular gene product has been in the dna E ts preparation and 15% in the wild-type prepara- isolated. For this reason it is essential that the complementing tion after 15 min. The remaining polymerase activity in the protein isolated from a ts mutant is thermolabile relative to heated dna E ts preparation (10%) was stimulated 3-fold by the wild-type protein as further evidence that the isolated 0.1 M KCl, suggesting the residual activity was DNA poly- protein is the product of that particular gene. As presented merase II (21). In both dna E complementation and DNA above, the dna G complementing activity purified from dna polymerase assays, mixtures of wild-type and dna E ts prepa- G ts cells is thermolabile when compared to wild-type dna G rations gave the sum of the inactivation curves of the indi- activity. In addition, temperature inactivation curves ob- vidual preparations. When the 1 M NaCl eluate from the tained by mixing dna G gene products isolated from the ts DNA-agarose column was chromatographed on phospho- mutant and wild-type were the sum of temperature inactiva- cellulose, two peaks of DNA polymerase III were detected, tion curves observed with each preparation. These results eluting at 0.11 M and 0.17 M potassium phosphate buffer support the conclusion that the protein which has been (pH 7.0). Both peaks were inhibited 90% by 0.1 M KCl. purified is the product of the dna G gene. DNA polymerase II activity eluted at 0.22 M phosphate The dna G gene product has a molecular weight of about buffer (pH 7.0). The dna E complementing activity followed 60,000, is N-ethylmaleimide-resistant, and binds poorly to the DNA polymerase III activity; there was no stimulation of single and double-stranded DNA. No enzymatic activity has dna E receptor crude extracts by the fractions containing been identified as yet which can be ascribed to the dna G DNA polymerase II.t protein. The protein did not catalyze incorporation of ribo- or deoxynucleotides nor did it~influence RNA or DNA synthesis catalyzed by purified DNA polymerases I, II, or III of E. coli. t Some preparations of purified DNA polymerase III did not stimulate dna E receptor crude extracts. We have found that the DNA-dependent and independent nucleoside triphosphatase lack of a quantitative relation between DNA polymerase III activities observed in glycerol-gradient fractions did not activity and dna E complementing activity was due to contami- cosediment with the dna G activity and were not more thermo- nation of DNA polymerase III preparations with stimulatory labile in preparations from dna G ts cells; thus, we conclude proteins that increased dNMP incorporation catalyzed by E. coli these activities are contaminants. DNA polymerases (22). Downloaded by guest on September 29, 2021 1618 Biochemistry: Wickner et al. Proc. Nat. Acad. Sci. USA 70 (1973) The complementation assay, as described, above, is also 7. Moses, R. & Richardson, C. C. (1970) Proc. Nat. Acad. Sci. being used to purify the dna B gene product. Partially USA 67, 674-681. 8. Schaller, H., Otto, B., Niisslein, V., Huf, J., Herrmann, R. & purified fractions, isolated from dna B ts cells, are thermolabile Bonhoeffer, F. (1972) J. Mol. Biol. 63, 183-200. compared to fractions isolated from dna C ts cells. Also by 9. Wickner, R. B. & Hurwitz, J. (1972) Biochem. Biophys. Res. this complementation assay, activities that stimulate dna C Commun. 47, 202-211. and dna D receptor crude extracts have been purified. Thus, 10. Wickner, W. T., Brutlag, D., Schekman, R. & Kornberg, A. by (1972) Proc. Nat. Acad. Sci. USA 69, 965-969. complementation in the 4X174 DNA-dependent system, it 11. Schekman, R., Wickner, W. T., Westergaard, O., Brutlag, should be possible to purify the products of all the known dna D., Geider, K., Bertch, L. L. & Kornberg, A. (1972) Proc. genes. Because a defined DNA template is used, it should be Nat. Acad. Sci. USA 69, 2691-2695. possible to analyze the mechanism of DNA synthesis carried 12. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. out by this system. (1972) Proc. Nat. Acad. Sci. USA 69, 3233-3237. 13. Nuisslein, V., Bonhoeffer, F., Klein, A. & Otto, B. (1972) in The Second Annual Harry Steenbock Symposium, eds. Wells, This work was supported by grants from the National Institute R. & Inman, R. (University Park Press, Maryland), in of Health (GM-13344) and the American Cancer Society (NP- press. 8900). S. W. is a Predoctoral trainee of The National Institute of 14. Campbell, J. L., Soll, L. & Richardson, C. C. (1972) Proc. Health, while M. W. is a Fellow of The Jane Coffin Childs Memo- Nat. Acad. Sci. USA 69, 2090-2094. rial Fund for Medical Research. 15. Sinsheimer, R. L. (1966) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper and 1. Hirota, Y., Ryter, A. & Jacob, F. (1968) Cold Spring Harbor Row, New York), pp. 569-576. Symp. Quant. Biol. 33, 677-693. 16. Franke, B. & Ray, D. S. (1970) Virology 44, 168-187. 2. Wechsler, J. A. & Gross, J. D. (1971) Mol. Gen. Genet. 113, 17. Bucher, T. (1947) Biochim. Biophys. Acta, 1, 292-314. 273-284. 18. Schaller, H., Nusslein, C., Bonhoeffer, F. J., Kurz, C. & 3. Carl, P. L. (1970) Mol. Gen. Genet. 109, 107-122. Neitzschmann, I. (1972) Eur. J. Biochen. 26, 474481. 4. Gefter, M. L., Hirota, Y., Kornberg, T., Wechsler, J. & 19. Sigal, N., Delius, H., Kornberg, T., Gefter, M. L. & Alberts, Barnoux, C. (1971) Proc. Nat. Acad. Sci. USA 68, 3150- B. (1972) Proc. Nat. Acad. Sci. USA 69, 3537-3541. 3153. 20. Jones, 0. W. & Berg, P. (1966) J. Mol. Biol. 22, 199-209. 5. Niisslein, V., Otto, B., Bonhoeffer, F. & Schaller, H. (1971) 21. Wickner, R. B., Ginsberg, B., Berkower, I. & Hurwitz, J. Nature New Biol. 234, 285-286. (1972) J. Biol. Chem. 247, 489497. 6. Fuchs, J. A., Karlstom, H. O., Warner, H. R. & Reichard, P. 22. Hurwitz, J., Wickner, S. & Wright, M. (1973) Biochem. (1972) Nature New Biol. 238, 69-71. Biophys. Res. Commun., 51, 257-267.

Correction. In the article "Structural and Functional Correction. In the article "Assembly of Rod-Shaped Virus Properties of Ribosomes Crosslinked with Dimethyl- In Vitro: Reconstitution with Cucumber Green Mottle suberimidate," by Slobin, L. I., which appeared in the Mosaic Virus Protein and Tobacco Mosaic Virus RNA," by Ohno, T., Inoue, H. & Okada, Y., which appeared in the December 1972 issue of Proc. Nat. Acad. Sci. USA 69, December 1972 issue of Proc. Nat. Acad. Sci. USA 69, 3769-3773, p. 3770, right-hand column, line 3 from top, 3680-3683, p. 3861, right-hand column, the first line should 10 mM KC1-6 mM 2-mercaptoethanol (pH 8.5) sh~uld read: "as 13 S, Fig. 2B); whereas. . . "; the fifth line should read: 100 mM KC1-6 mM 2-mercaptoethanol (pH 8.5). read: "(Fig. 2A). The results. ". Downloaded by guest on September 29, 2021