Proc. Natl. Acad. Sci. USA Vol. 74, No. 5, pp. 1937-1941, May 1977 Enzymatic initiation of DNA synthesis by yeast DNA polymerases (DNA replication/yeast RNA nucleotidyltransferases/RNA-DNA linked molecules) P. PLEVANI* AND L. M. S. CHANG Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 Communicated by R. B. Setlow, February 28, 1977

ABSTRACI Partially purified yeast RNA polymerases (RNA stimulation with synthesis initiated by RNA polymerase I nucleotidyltransferases) initiate DNA synthesis by yeast DNA compared with that initiated by RNA polymerases II and polymerase (DNA nucleotidyltransferase) I and to a lesser extent III. yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimu- MATERIALS AND METHODS lated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase Materials. Deoxynucleoside triphosphates (dNTPs) were initiation of the DNA polymerase II reaction. RNA polymerases prepared as described (15). Phage fd DNA was generously I and III are more efficient in initiation of DNA synthesis than supplied by R. Benzinger, University of Virginia. Commercial RNA polymerase II. Analyses of the products of fd DNA repli- sources of other materials were as follows: rNTPs from cation show noncovalent linkage between the newly synthesized Schwarz/Mann, radioactive from New England DNA and the template DNA, and covalent linkage between the Nuclear, a-amanitin from Sigma Chemical Co., bovine serum newly synthesized RNA and DNA. albumin and QB RNA from Miles Laboratories, and calf thymus DNA polymerases (DNA nucleotidyltransferases) from DNA from Worthington Biochemical Corp. prokaryotic and eukaryotic cells do not initiate chains de novo Yeast Cells. Saccharomyces cerevisiae, strain D 278-1OB (1, 2). In the replication of single-stranded coliphage DNA (pet[p+]) (16), was grown to late logarithmic phase, harvested, templates in vitro, a rifampicin-resistant RNA polymerase and stored as described (12). (RNA nucleotidyltransferase) is believed to be required for the Enzymes. Yeast DNA polymerases I and II were purified as conversion to double-stranded replicative form for kX174 and described (12). Yeast RNA polymerase I, II, and III were pu- G4 DNAs (3). The conversion of phage M13 DNA depends on rified from 60 g of frozen cells. Frozen cells were lysed in an a rifampicin-sensitive RNA polymerase (3). The enzymatic Eaton press at 9000 lb/inch2 (62 MPa) (17) and suspended in replication of phage T4 and T7 DNA templates is stimulated equal volume of 0.7 M (NH4)2SO4, 1 mM phenylmethylsul- X by the presence of rNTPs, although RNA polymerase activity fonylfluoride, 1% dimethyl sulfoxide in buffer A [50 mM is not required (4, 5). Ribonucleotide initiation of DNA synthesis Tris-HCI, pH 7.9/0.5 mM dithiothreitol/0.1 mM EDTA/10% had been implicated in the replication of polyoma DNA in (wt/vol) glycerol]. The lysate was treated for 30 sec at 100 W isolated nuclei (6), slime molds (7), and mammalian cells (8). in a Branson Sonifier to reduce the viscosity, and was then In the replication of synthetic DNA templates, DNA poly- clarified by centrifugation for 20 min at 10,000 X g. Nucleic merase-a from calf thymus uses oligoribonucleotide initiators acids in the clarified extract were removed by precipitation while DNA polymerase-,B does not use oligoribonucleotides with 0.5% protamine sulfate followed by centrifugation. The efficiently (9). On single-stranded HeLa DNA, RNA poly- protamine sulfate supernatant was diluted with two volumes merase from Escherichia coli initiates DNA synthesis catalyzed of buffer A and adsorbed onto a DEAE-Sephadex A-25 column. by HeLa cell DNA polymerase-a, but not by DNA polymer- RNA polymerases were eluted with a linear gradient of ases-f and --y (10). Eukaryotic enzymes capable of initiating (NH42S04 from 0.05-0.425 M. Active fractions were pooled DNA synthesis have not been demonstrated. for RNA polymerases I, II, and III, and the protein in each pool Two DNA polymerases are present in yeast (11, 12). The was precipitated by dialysis against 90% saturated (NH4)2SO4 properties of yeast DNA polymerase I are similar to those of in buffer A. The precipitates were collected by centrifugation, mammalian DNA polymerase-a. DNA polymerase II is similar redissolved in 50 mM Tris-HCI, pH 7.9/0.1 mM EDTA/0.5 mM to prokaryotic DNA polymerases in that it has an associated dithiothreitol/50% (wt/vol) glycerol, and stored at -20°. 3'-exonuclease (11-14). Experiments using synthetic DNA Enzyme Assays. DNA polymerase activities were assayed templates showed that DNA polymerase I readily accepts an as described (12). One DNA polymerase unit is 1 nmol of total oligoribonucleotide initiator while DNA polymerase II does not polymerized into DNA per hr. RNA polymerase (12). The availability of yeast RNA polymerases permits in- activities were assayed essentially as described (18) except that vestigation of enzymatic initiation of DNA synthesis in a ho- 0.028 mM radioactive UTP was used in all assays. One RNA mologous system. The results reported here, using a single- polymerase unit corresponds to incorporation of 1 nmol of UMP stranded circular DNA template, show that efficient enzymatic into RNA in 10 min. The reaction mixture used for DNA initiation occurs only with DNA polymerase I. All three yeast polymerase and RNA polymerase coupled reactions contained RNA polymerases can initiate synthesis on the fd DNA template 50 mM Tris-HCI, pH 7.9/0.5 mM dithiothreitol/8 mM in the absence of rNTPs. The presence of rNTPs gives greater MgCl2/1 mM MnCl2/50 ,ug of bovine serum albumin per ml/0.1 mM each of dCTP, dGTP, dATP, and [methyl-3H]- in the or absence of 0.1 Abbreviations: Those of IUPAC-IUB Commission on Biochemical dTTP (360-750 cpm/pmol), presence Nomenclature are used; buffer A, 50 mM Tris-HCl, pH 7.9/0.5 mM mM each CTP, UTP, GTP, and [2-]4C]ATP (29 cpm/pmol)/ dithiothreitol/0.1 mM EDTA/10% (wt/vol) glycerol. 150 ,g of native or heat-denatured calf thymus DNA per ml * On leave from Istituto di Biologia Generale, E.U.L.O. Brescia, or 25 tg of fd DNA per ml. The coupled reactions were gen- Italy. erally carried out in 115 ,ul, and the enzymes were added to the 1937 Downloaded by guest on September 29, 2021 1938 Biochemistry: Plevani and Chang Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Initiation of DNA synthesis catalyzed by yeast DNA polymerases by yeast RNA polymerases on heat-denatured calf thymus DNA

I Relative stimulation of

z 0 DNA DNA PI-~~~~~~~~~~~I0 RNA polymerase polymerase polymerase Nucleotides I II -0.3 - ~~~0 dNTPs, rNTP (41) (2.3) 2- -~~~~~~~~~~~0.2_ I (boiled) dNTPs, rNTPs 1.0 (92) 1.0 (2.4) dNTPs 3.8 2.7 -01 Z I I dNTPs, rNTPs 5.8 2.7 dNTPs, rNTPs 1.0 (78) 1.0 (2.2) 4b 6'0 8,0 1 00 20 II (boiled) II dNTPs 3.6 1.7 FRACTION NUMBER II dNTPs, rNTPs 5.0 1.8 FIG. 1. Chromatography of yeast RNA polymerases on DEAE- III (boiled) dNTPs, rNTPs 1.0 (68) 1.0 (2.1) Sephadex A-25. The diluted protamine sulfate supernatant was III dNTPs 5 1.7 loaded onto a DEAE-Sephadex A-25 column (4.5 X 27 cm) that had III dNTPs, rNTPs 6.6 1.5 been equilibrated with 0.05 M (NH4)2SO4 in buffer A. The column was washed with one column volume of the same buffer and protein Reactions were for 1 hr (see Materials and Methods). The amounts was eluted with a 3-liter linear gradient of (NH4)2SO4 from 0.05 to of DNA polymerase present were 10 units and 0.4 unit for DNA 0.425 M. Fractions (22 ml each) were collected and 50-,Al aliquots were polymerases I and II, respectively. The amounts of RNA polymerase used to assay for RNA polymerase activity. About 90% of the total present were 0.07, 0.14, and 0.02 unit for RNA polymerases 1, I, and enzyme activity present in the crude extract was recovered in the III, respectively. The control reactions contained equal volumes of column fractions. RNA polymerase solution except that the enzyme solution was heated for 5 min in a boiling-water bath. The results are normalized to the control values. The numbers in parentheses are pmol of [3H]dTMP reaction mixtures in an ice bath. To start the reactions, tubes polymerized per hr. The amounts of [14C]AMP polymerized were 35, were transferred to a 350 water bath. Samples (10-25 ,1) were 290, and 47 pmol in reactions containing DNA polymnerase I and taken at various times and processed for acid-insoluble materials rNTPs, and 32, 79, and 26 pmol in reactions containing DNA poly- on glass fiber disks (19). The disks were analyzed for 14C (RNA merase II and rNTPs for RNA polymerases I, II, and III, respective- product) and 3H (DNA product) by liquid scintillation count- ly. ing. Analysis of Products of fd DNA Replication. The reactions presence of yeast RNA polymerase in the presence or absence with fd DNA were done as described and terminated by ad- of rNTPs. The polymerization of ribonucleotides was readily dition of EDTA, NaCl, and NaOH to final concentrations of demonstrated in these reactions (data not shown). 50 mM, 0.1 M, and 0.3 M, respectively, and incubation at 350 When heat-denatured calf thymus DNA is used as template, for 15 min. Alkaline sucrose gradients were 5-20% (wt/wt) RNA polymerases stimulate DNA polymerase activities 4- to sucrose in 1 mM EDTA, 0.1 M NaCl, and 0.1 M NaOH, and 7-fold. All three RNA polymerases stimulated DNA polymerase centrifugation was in a SW 50.1 rotor for 6 hr at 45,000 rpm at I in the absence of rNTPs (Table 1). Further stimulation is seen 40. The products were analyzed on formaldehyde/CsCl/ in the presence of all four rNTPs. A modest stimulation of DNA Cs2SO4 gradients essentially as described (10). polymerase II reactions by RNA polymerases is also noted, but this stimulation cannot be increased by addition of rNTPs and RESULTS will not be considered further in this discussion. Separation of RNA polymerases from yeast The stimulation of DNA polymerase by RNA polymerases acting on the denatured DNA template in the absence of rNTPs RNA polymerase activities were separated into four peaks by is probably not due to the presence of oligonucleotide con- chromatography on DEAE-Sephadex A-25 (Fig. 1). The three taminants in the enzyme fractions since the control reactions major peaks of enzyme activities correspond to RNA poly- contained appropriate amounts of boiled enzymes.t The ad- merases I, II, and III by comparison of their properties with ditional stimulation of DNA polymerase I activity by rNTPs those described from several laboratories (20-22). The prop- requires the presence of all four rNTPs. Elimination of any of erties examined include divalent cation requirements, salt de- the four rNTPs resulted in rates identical to those observed in pendence, template specificities, and a-amanitin sensitivi- the absence of all four rNTPs (data not shown). ties. Stimulation of yeast DNA polymerase activity by yeast t The boiled RNA polymerase fractions do not inhibit the DNA RNA polymerases polymerase reactions. The 2-fold stimulation observed with DNA polymerase I reactions by the boiled RNA polymerases can probably Since yeast DNA polymerases use native or heat-denatured be explained by the presence of (NH4)2SO4 in the boiled enzymes DNA as templates very poorly (12), we have used these tem- since 60 mM (NH4)2SO4 stimulates yeast DNA polymerase I activity plates to test for the ability of the yeast RNA polymerases to by 2-fold (12). DNA polymerase II activity is not stimulated at the initiate and stimulate enzymatic DNA synthesis. All of the re- same concentration of (NH4)2SO4. The possibility that RNA poly- merase is required for the binding of contaminating oligonucleotides actions described in this communication contained exogenous to DNA was examined by the addition of boiled enzyme to a coupled DNA template. In the absence of added DNA, no incorporation reaction of homogeneous yeast RNA polymerase I and yeast DNA of dNTPs or rNTPs can be detected (data not shown). When polymerase I. The boiled enzyme did not significantly stimulate (less native calf thymus DNA was used as template, no stimulation than 40%) the enzymatic DNA synthesis initiated by homogeneous of DNA synthesis by either DNA polymerase was caused by the RNA polymerase I (data not shown). Downloaded by guest on September 29, 2021 Biochemistry: Plevani and Chang Proc. Natl. Acad. Sci. USA 74 (1977) 1939

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0 30 60 0 30 60 0 30 60 REACTION TIME (MIN) FIG. 2. Initiation of DNA synthesis catalyzed by yeast DNA polymerase I by yeast RNA polymerases on fd DNA template. The coupled reactions were done as described in Materials and Methods. Each reaction mixture contained 10 units of DNA polymerase and 0.05,0.09, and 0.11 unit of RNA polymerases I (A), 11 (B), and III (C), respectively. (A) RNA polymerase + rNTP; (-) RNA polymerase; (0) boiled RNA polymerase. Initiation of DNA synthesis by yeast RNA polymerases RNA polymerase II and 300-1000lg/ml for RNA polymerase on fd DNA template I (21, 22). RNA polymerase III from yeast is not inhibited by To simplify analysis of the initiation event we chose single- a-amanitin (21, 22). We examined the effect of 50 and 1500 stranded circular fd DNA for further studies. Time courses for ,g of a-amanitin per ml on the RNA polymerase stimulation synthesis on fd DNA template catalyzed by DNA polymerase of DNA polymerase I (Table 2). Initiation of DNA synthesis by I and stimulated by RNA polymerases in the presence and ab- all three RNA polymerases in the absence of rNTPs is not sig- sence of rNTPs are presented in Fig. 2. All three RNA poly- nificantly affected at the low concentration of a-amanitin but merases stimulate DNA synthesis on fd DNA in the absence of is inhibited 20-40% at the high concentration. The inhibition rNTPs, and this stimulation is enhanced by the presence of all patterns for the rNTP-stimulated initiation are more complex. four rNTPs. When the results are normalized to equal amounts For RNA polymerase I, inhibited only at high concentration of ribonucleotide polymerization (insets in Fig. 2), RNA poly- of a-amanitin, the stimulation of fd DNA synthesis by DNA merase I and RNA polymerase III stimulate fd DNA replication polymerase is accordingly not reduced at low concentration but more efficiently than RNA polymerase II. is greatly reduced at the high concentration of a-amanitin. For A lag phase is observed for DNA polymerase reactions RNA polymerase II, at 50 ,ug of a-amanitin per ml, RNA syn- stimulated by addition of RNA polymerase. Incubation of the thesis is reduced by more than 95% but rNTP-stimulated DNA fd template and substrates with RNA polymerase prior to the synthesis is reduced only by about 3. The RNA polymerase II addition of DNA polymerase eliminates the lag phase (data not stimulation in the presence of rNTPs is virtually abolished at shown). These results suggest that the RNA polymerases pro- the high concentration of a-amanitin. Although ribonucleotide duce some kind of initiator for the DNA polymerase. polymerization by RNA polymerase III is not inhibited by a- amanitin (21, 22), the stimulation produced in the presence of Effects of a-amanitin on initiation of DNA synthesis rNTPs is reduced by about 50% at the low concentration of by yeast RNA polymerases a-amanitin and further reduced at the high concentration. Yeast RNA polymerases exhibit different sensitivities to a- The pattern of a-amanitin inhibition is consistent with known amanitin (21, 22). The concentration of a-amanitin required properties of RNA polymerase 1 (21, 22). The partial inhibition for 50% inhibition of enzyme activity is about 10 ,ug/ml for of DNA synthesis stimulated by RNA polymerase II in the

Table 2. Inhibition of initiation of DNA synthesis by yeast RNA polymerases on fd DNA by a-amanitin RNA polymerase I RNA polymerase II RNA polymerase III ce-Amanitin (4glml) Nucleotides [3H]dTMP ['4C]AMP [3H]dTMP [14C]AMP [3H]dTMP ['4C]AMP dNTPs, rNTPs 8.7* 0 8.8* 0 6.4* 0 dNTPs 132 77 78 - dNTPs, rNTPs 484 230 212 525 175 60 50 dNTPs 125 79 70 50 dNTPs, rNTPs 516 208 169 20 123 46 1500 dNTPs 86 63 44 1500 dNTPs, rNTPs 165 56 78 7 75 49 Results are given in pmol of nucleotide polymerized per reaction per hr. * Control reactions contained boiled RNA polymerases. Reactions were done as described in Materials and Methods. The amounts ofenzymes used were the same as described in the legend to Fig. 2. Downloaded by guest on September 29, 2021 1940 Biochemistry: Plevani and Chang Proc. Natl. Acad. Sci. USA 74 (1977)

0 1.0- 10- Xwu- 1 ~~~~~~~1.8t- 0 zZU 60o 1.7 U_ 0 0.5- 5- 40Q -4 E -o~~~~~~~~~~~~~~~- ~~~~~~~~~~0-~Z~~~~~~5 E CL 20 -2 0 10 20 30 40 50 0 10 20 30 40 50 T FRACTION NUMBER S FIG. 3. Alkaline sucrose gradient analyses of the products of fd DNA replication initiated by RNA polymerase I. Reactions were 10 20 30 40 50 carried out with or without the presence of rNTPs, terminated, and FRACTION NUMBER analyzed on an alkaline sucrose gradient as described in Materials FIG. 4. Analysis of the products of fd DNA replication initiated and Methods. Q, top of gradient; (), bottom of gradient. fd DNA by RNA polymerase I on a formaldehyde/CsCI/Cs2SO4 gradient. The marker positions are shown. (0) dNTPs + rNTPs; (0) dNTPs only. reaction was carried to 40% replication in the presence of RNA (A) Early products of replication; (B) products of more extensive polymerase I, DNA polymerase I, fd DNA, dNTPs, and rNTPs as synthesis. described in the legend to Fig. 1 and Materials and Methods. The reaction was terminated and the products analyzed as described (10). presence of rNTPs at the low concentration of a-amanitin can QB RNA and fd DNA marker positions were obtained from a separate be explained by the residual ribonucleotide polymerizing ac- gradient by absorbance measurements at 260 nm. The tivity (about 4%). This residual amount of RNA synthesis could markers were treated with formaldehyde at 900 in the same way as provide the DNA polymerase with the initiator required for the sample. polymerization of dNTPs. The a-amanitin inhibition of reac- proceed to 12.5% in the presence of rNTPs and 7.5% in the tions initiated by RNA polymerase III is anomalous since RNA absence of rNTPs, the average size of the products increased polymerase III is not sensitive to a-amanitin (21, 22). Never- to 8.2 S and 10.7 S, respectively. Degradation of the template theless, the stimulation of DNA synthesis in the presence of DNA probably does not occur since incubation of fd DNA with rNTPs is inhibited. The a-amanitin inhibition and an analysis enzymes in the absence of substrates for over 1 hr did not pro- of the amounts of ribonucleotides polymerized in these reactions duce any observable alteration of fd DNA on an alkaline sucrose does not permit us to assign the stimulation by RNA polymerase gradient. The results of this analysis suggest initiation of new III in the presence of rNTPs to contamination with RNA chains in DNA polymerase reactions stimulated by yeast RNA polymerases I and II. The rNTP stimulation of RNA polymerase polymerase. III could be due to the presence of an a-amanitin-sensitive protein that is not required for RNA synthesis but that does Analysis of products of ribonucleotide-initiated fd stimulate DNA synthesis. DNA replication by formaldehyde/CsCl/Cs2SO4 gradient Analysis of products of fd DNA replication on alkaline Although the initiation of fd DNA synthesis by yeast RNA sucrose gradients polymerases is not absolutely dependent on the presence of The results presented demonstrate that yeast RNA polymerases rNTPs, extensive stimulation is observed when rNTPs are can stimulate fd DNA synthesis catalyzed by yeast DNA present in the reactions. It is of interest to know if the RNA polymerase I in the presence or absence of rNTPs. The lack of synthesized by RNA polymerases is used as initiator for DNA total dependence on the presence of rNTPs in the initiation of synthesis by DNA polymerase. The products of fd DNA syn- DNA synthesis had been observed in the replication of coli- thesis initiated by yeast RNA polymerase I and catalyzed by phage DNA in vitro (4, 5). In the replication of phage T4 DNA yeast DNA polymerase I in the presence of rNTPs were ana- in vitro, the initial products synthesized in the absence of rNTPs lyzed on a formaldehyde/CsCl/Cs2SO4 gradient (Fig. 4). The are covalently linked to the template DNA while the products results show that the bulk of the RNA synthesized has a density are not covalently linked to the template in the presence of lower than Qf RNA but higher than fd DNA. Since we have rNTPs (5). In order to examine whether similar mechanisms not analyzed the sizes of the products throughout the gradient, are also operating in the yeast system in vitro, we have analyzed we are not able to comment on the precise composition of these the products of fd DNA replication on alkaline sucrose gradi- materials. The fact that most of the RNA products banded at ents. Fig. 3 shows the alkaline sucrose gradient patterns of the densities lighter than the marker RNA suggests that most of the products of fd DNA synthesis initiated by RNA polymerase I RNA chains synthesized have DNA covalently attached. The and catalyzed by DNA polymerase I in the presence and the bulk of the newly synthesized DNA, however, sedimented near absence of rNTPs. Fig. 3 A shows the results obtained from the density of fd DNA, and in these fractions there was an av- products at early stages of DNA replication, 0.8% replication erage ratio of deoxynucleotides to ribonucleotides of about 25. in the presence of rNTPs and 0.3% replication in the absence Although some of the newly synthesized DNA products can be of rNTPs. The products are not covalently linked to the tem- expected to have no ribonucleotide at the 5'-termini, the high plate DNA. The average sedimentation coefficients of the deoxynucleotide to ribonucleotide ratio in the bulk of the DNA products are about 5.7 S in the presence of rNTPs and 6.9 S in products indicates that most of the RNA initiators used by DNA the absence of rNTPs. When the replication was allowed to polymerase are of relatively short chain length. Downloaded by guest on September 29, 2021 Biochemistry: Plevani and Chang Proc. Natl. Acad. Sci. USA 74 (1977) 1941 DISCUSSION for nucleolar DNA replication. RNA polymerase II in the nu- Yeast DNA polymerases are unable to initiate new chains (12). cleoplasm (25) could effect a mixture of deoxynucleotide and The experiments presented show that all three yeast RNA ribonucleotide initiation. The participation of these hypothetical polymerases can initiate new chains on circular single-stranded mechanisms for initiation of yeast DNA polymerases in vivo fd DNA in concert with DNA polymerase I in the absence of may be susceptible to further analysis using yeast mutants (26, rNTPs. The mechanism for the chain initiation catalyzed by 27). RNA polymerase in the absence of rNTPs is unusual, and not This research was supported by U.S. Public Health Service Research all molecular details are known. In contrast to the replication Grant CA 17770.from the National Cancer Institute. of fX174 DNA by E. coli DNA polymerase I (23), the initiation The costs of publication of this article were defrayed in part by the of fd DNA by yeast RNA polymerases examined in our exper- payment of page charges from funds made available to support the iments is not due to oligonucleotide contamination of the en- research which is the subject of the article. This article must therefore zymes since boiled enzymes were used in the control reactions. be hereby marked "advertisement" in accordance with 18 U. S. C. In the absence of rNTPs, the newly synthesized DNA in the §1734 solely to indicate this fact. yeast system is not covalently linked to the template as is ob- served in the replication of phage T4 DNA in vitro (5). The 1. Kornberg, T. & Kornberg, A. (1974) in The Enzymes, ed. Boyer, possibility that template fd DNA was degraded and used as an P. D. (Academic Press, New York), Vol. 10, pp. 119-144. initiator appears unlikely since incubation of fd DNA with 2. Bollum, F. J. (1975) in Progress in Nucleic Acid Research and enzymes in the absence of substrates did not produce any al- Molecular Biology, ed. Davidson, J. N. & Cohn, W. E. (Academic teration of fd DNA structure. The results are compatible with Press, New York), Vol. 15, pp. 109-144. 3. Schekman, R., Weiner, A. & Kornberg, A. (1974) Science 186, the hypothesis that yeast RNA polymerases or factors inter- 987-993. acting with RNA polymerases can utilize dNTPs to initiate new 4. Hinkle, D. C. & Richardson, C. C. (1975) J. Biol. Chem. 250, chains. If this is true, the chains produced must be small, since 5523-5529. no acid-precipitable products can be detected in the RNA 5. Morris, C. F., Sinha, N. K. & Alberts, B. M. (1975) Proc. Natl. polymerase reactions (unpublished results). If this mechanism Acad. Sci. USA 72,4800-4804. for chain initiation occurs in vivo, it would not be detected by 6. Reichard, P., Eliasson, R. & Sbderman, G. (1974) Proc. Natl. the current techniques of analysis (6-8). The hypothesis that Acad. Sci. USA 71,4901-4905. yeast RNA polymerases can produce short oligodeoxynucleo- 7. Waqar, M. A. & Huberman, J. A. (1975) Cell 6,551-557. tides on a DNA template deserves further investigation. 8. Waqar, M. A. & Huberman, J. A. (1975) Biochim. Biophys. Acta Ribonucleotide initiation is readily demonstrated with yeast 383,410-420. 9. Chang, L. M. S. & Bollum, F. J. (1972) Biochem. Biophys. Res. DNA polymerase I, but not with DNA polymerase II. Amongst Commun. 46, 1354-1360. the three RNA polymerases, RNA polymerase I is most efficient 10. Spadari, S. & Weissbach, A. (1975) Proc. Natl. Acad. Sci. USA in ribonucleotide initiation on fd DNA. Partial sensitivity of 72,503-507. rNTP stimulation of initiation by RNA polymera'se II at low 11. Wintersberger, N. & Wintersberger, E. (1970) Eur. J. Biochem. concentration of a-amanitin suggests that RNA polymerase II 13, 11-19. can also provide the RNA initiator for DNA polymerase I. The 12. Chang, L. M. S. (1977) J. Biol. Chem., in press. inhibition of RNA polymerase III initiation in the presence of 13. Helfman, W. B. (1973) Eur. J. Biochem. 32,42-50. rNTPs by a-amanitin suggests the involvement of a factor that 14. Wintersberger, E. (1974) Eur. J. Biochem. 50, 41-47. is not required for RNA synthesis. Our preliminary results show 15. Chang, L. M. S. & Bollum, F. J. (1971) J. Biol. Chem. 246, that RNA yeast (18) initiates 909-916. homogeneous polymerase I from 16. Sherman, F. (1965) in Regulations chez les Microorganism, Coll. DNA synthesis by DNA polymerase I only in the presence of Int. Centre Nat. Rech. Su, Marseille, July 23-27, 1963 (Editions all four rNTPs (unpublished results). To explain this observa- du C.N.R.S., Paris), p. 465. tion, it is necessary to propose that a factor capable of inter- 17. Bhargava, M. M. & Halvorson, H. 0. (1971) J. Cell. Biol. 49, acting with RNA polymerase I is required for the initiation of 423-427. DNA synthesis in the absence of rNTPs and that this factor is 18. Valenzuela, P., Weinberg, F., Bell, G. & Rutter, W. J. (1976) J. removed from RNA polymerase I during more extensive pu- Biol. Chem. 251, 1464-1470. rification (18). In view of the fact that all three yeast RNA 19. Bollum, F. J. (1966) in Procedures in Nucleic Acid Research, eds. polymerases can initiate enzymatic DNA synthesis, it is possible Cantoni, G. & Davies, D. (Harper and Row, New York), to speculate that a DNA initiation factor capable of interacting pp. 296-300. RNA 20. Adam, R., Schultz, L. & Hall, B. (1972) Proc. Natl. Acad. Sci. USA with all three polymerases is present in the cell. 69, 1702-1706. It is possible that chain initiation in DNA synthesis in eu- 21. Schultz, L. D. & Hall, B. D. (1976) Proc. Natl. Acad. Sci. USA karyotes occurs by more than one mechanism. The rolling 73, 1029-1033. hairpin model described by Tattersall and Ward (24) for par- 22. Hager, G., Holland, M., Valenzuela, P., Weinberg, F. & Rutter, voviruses uses preformed 3'-hydroxyls on a palindromic DNA W. J. (1976) in RNA Polymerase, eds. Losick, R. & Chamberlain, sequence for initiation and completion of DNA chains. DNA M. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), of polyoma virus replicated in nuclei has RNA and DNA joints pp. 745-762. (6), implying DNA initiation on the 3'-hydroxyl of RNA chains. 23. Goulian, M. & Kornberg, A. (1967) Proc. Natl. Acad. Sci. USA The present study resurrects the possibility that some form of 58, 1723-1730. de novo initiation may occur, 24. Tattersall, P. & Ward, D. (1976) Nature 263, 106-109. by deoxynucleoside triphosphates 25. Roeder, R. G. & Rutter, W. J. (1970) Proc. Natl. Acad. Sci. USA although not mediated by DNA polymerase alone. If the results 65,675-682. in vitro described here reflect the situation in vivo, one might 26. Hartwell, L. H., Mortimer, R. K., Culotti, T. & Culotti, M. (1973) postulate that RNA polymerase I (shown to be most effective Genetics- 74, 267-286. in RNA initiation, possibly with factors) present in the nucleolus 27. Hereford, L. M. & Hartwell, L. H. (1974) J. Mol. Biol. 84, (25) might cause a predominance of ribonucleotide initiation 445-462. Downloaded by guest on September 29, 2021