RNA primers in simian virus 40 DNA replication

J. Mol. Biol. (1977) 116, 549-567

RNA Primers in Simian Virus 40 DNA Replication II?. Distribution

of 5’ Terminal Oligoribonucleotides

GABRIEL KAUFMANN~,

in Nascent DNA

STEPHEN ANDERSON AND MELVIN L. DEPAMFHIZLIS

Department of Biological Chemistry, Harvard Medical School Boston, Mass. 02115, U.S.A. (Received 31 January 1977, and in revised form 24 April 1977) A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identiCed by transfer of a fraction of the 32P from the 5’ position of a deoxyribonucleotide to 2’( 3’)rNMPs upon either alkaline hydrolysis or RNAase Tz digestion of SV40 replicating [32P]DNA. Alkaline hydrolysis also exposed 5’ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with Pl nuclease. The RNADNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5’ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a decked length in the presence of either RNAase T, or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least sevenresidues. Since alkaline hydrolysis of similar Okazaki pieces revealed that onethird to one-half of them contained rN-3aP-dN linkages, the oligoribonucleotides must be covalently attached to the 5’ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers. Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5’ end of growing daughter strands. 32P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII+III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences. t Paper I in this series is Anderson, et al. (1977). $ Present address: Department of Biochemistry, The Weizmann Institute of Science, P.O. Box 26, Rehovot, Israel. 649

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We propose a model in which a deficiency of one to two double helical turns per nucleosomo in the unreplicated DNA provides a periodic, nucleotide sequence-independent, initiation signal for the synthesis of Okazaki pieces. This signal is exposed upon dissociation of the nucleosome during DNA replication.

1. Introduction During DNA replication, daughter strands are initiated at the origins of replicons. More DNA chain initiation events are thought to occur as the replication forks advance. This was suggested by the following argument. DNA chains grow only in the 5’ to 3’ direction. If both strands are synthesized independently and simultaneously then the strand growing against the fork movement (the retrograde strand) must be synthesized discontinuously. In numerous eukaryotic and prokaryotic DNA replication systems, DNA precursors appear first in short chains (Okazaki pieces) which are later incorporated into longer chains. In some of these studies Okazaki pieces were found on both arms of the replication fork (Kornberg, A., 1974; Gefter, 1975; Edenberg & Huberman, 1975). DNA polymerases require a 3’-OH terminated oligonucleotide primer to initiate synthesis, but RNA polymerases do not. DNA polymerases catalyze a covalent attachment of DNA to either an RNA or DNA primer in vitro (Wells et d., 1972). In the case of coliphage M13, Esche&hia coli RNA polymerase (Brutlag et aZ., 1972) and RNA synthesis (Wickner et al., 1973) were found necessary for DNA replication. These facts underlie the “RNA priming hypothesis” (Brutlag et al., 1972; Wells et al., 1972; Sugino et al., 1972). According to this hypothesis, transient RNA chains serve to initiate DNA synthesis at the origins of replicons and Okazaki pieces. The transcription of an RNA primer at the origin of DNA replication has been reported for the single-stranded DNA coliphage G4 (Rowen & Bouche, 1976). A similar RNA primer has been found at the origin of DNA replication in the doublestranded DNA plasmid colicin El (Helinski, 1976). RNA priming of Okazaki pieces is supported mainly by the presence of RNADNA linkages in nascent. DNA chains. The best documented analysis monitoring these linkages measures the transfer of 32P from precursor [c+~~P]~NTP to 2’(3’) rNMPs upon incubation of nascent DNA under alkaline conditions. RNA-DNA linkages were reported in replicating DNA from many organisms : polyoma (Magnusson et al., 1973; Hunter & Francke, 1974; Pigiet et al., 1974; Sadof & Cheevers, 1973), hamster cells (Waqar & Huberman, 1975b), human lymphocytes (Tseng & Goulian, 1975), Physarum polycephalum (Waqar & Huberman, 1975a) and E. coli (Sugino et al., 1972; Sugino $ Okazaki, 1973; Hirose et al., 1973; Kurosawa et al., 1975). In some of these studies, the ratio of RNA-DNA linkages to the number of Okazaki pieces was reported to be unity (cf. Waqar & Huberman, 19756). Other observations consistent with the notion of RNA priming of Okazaki pieces are : (1) Okazaki pieces band in a cesium sulfate equilibrium gradient at a density greater than that of single-stranded DNA, and shift to the density of single-stranded DNA upon incubation in alkali (Hunter t Francke, 1974) ; (2) incubation of nascent DNA under alkaline conditions exposes 5’-OH termini (Pigiet et al., 1974; Hirose et al., 1973; Kurosawa et al., 1975); (3) an RNA chain of about ten nucleotides covalently linked to nascent DNA was isolated from polyoma replicative intermediates (Reichard et al., 1974; Elliasson et al., 1974). In order to understand the relation between these RNA-DNA linkages and the

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551

process of DNA replication, we began studies on their occurrence and dynamics in replicating simian virus 40 (SV40) DNA. We have used a cell-free system consisting of nuclei isolated from infected monkey kidney cells, and cytosol (DePamphilis & Berg, 1975; DePamphilis et aZ., 1975). Cytosol, the 100,000 g supernatant from cell cytoplasm, contains replication factors thought to have leaked out of the nucleus. This system faithfully converts SV4O(RI) DNA (the replicating form) into mature SV40 covalently closed, superhelical DNA. It is therefore expected that intermediate steps in the replication process are faithfully performed as well. In this paper we report the occurrence of an oligoribonucleotide of at least seven residues covalently attached to the 5’ end of a fraction of Okazaki pieces. This fraction appeared uniformly distributed among the various lengths of Okazaki pieces and throughout the SV40 genome. In a separate paper, we critically demonstrate that all 16 possible RNA-DNA covalent linkages occur in SV40 replicating DNA at frequencies predicted from a nearest-neighbor analysis of SV40 DNA (Anderson et al., 1977). These linkages were removed at the same rate that Okazaki pieces were joined to the growing strands of nascent DNA.

2. Materials and Methods (a) Cells and vim9

All experiments were done with a CV- 1 African

green monkey kidney cell line obtained from P. Tegtmeyer. Cells were routinely grown at 37°C in loo-mm diam. Lux dishes using Dulbecco’s modified Eagle’s medium, supplemented with 5% (v/v) foetal calf serum. Penicillin G (500 units/ml) and streptomycin .sulfate (100 rg/ml) were added to virus-infected cells as a safeguard against bacterial contamination. The small plaque SV40 strain Rh911 (Girardi, 1965) was grown by infection of MA134 cells (Microbiological Associates) at 0.01 or less plaque-forming units/cell. Virus was extracted 10 to 12 days later by scraping the infected cells into their medium with a rubber policeman and subjecting the suspension to 3 cycles of freezing in a solid CO,/ethanol bath and thawing at 30°C. The cellular debris were removed by sedimentation at 10,000 g for 10 min. The supernatant was adjusted to 10% m foetal calf serum before being stored in small portions at -35%. (b) Preparation

of cytoaol

Small amounts of cytosol were prepared from uninfected CV-1 cells as described by DePamphilis & Berg (1975). All steps were carried out at 2°C or on ice. Large amounts were prepared from 100 roller bottles by decanting the medium, washing the cells twice with 25 ml of buffer A, and removing the cells with a rubber policeman. This resulted in a lysate which was centrifuged for 10 min at 5000 g to remove the nuclei. The nuclear pellet was suspended in 200 ml of buffer A, homogenized by 3 strokes of a loose-fitting (pestle A) Dounce homogenizer and again sedimented. The 2 supernatants were combined, adjusted to 0.1 M-KCl, and centrifuged at 100,000 g for 1 h. The soluble cytosol fraction was brought to 80% saturation with solid ammonium sulfate, and the precipitate was collected by centrifugation, resuspended and dialyzed overnight against 10 m&r-HEPES-Na (pH 7.8), 50 IIIM-KCl, 6.5 mm-MgCl,, 0.5 m&r-dithiothreitol, 0.4 M-ethylene glycol. The cytosol fraction contained 10 to 15 mg protein/ml and was stable for at least 2 months at - 70°C. (c) XV40

DNA

synthesis

in kolated

nuclei

Nuclei were isolated from SV40-infected CV-1 cells as follows. Cells were infected as previously described (DePamphilis & Berg, 1975; DePamphilis et al., 1975). The medium was removed from the dishes 36 h after infection and the cells washed twice with 10 ml of buffer A (all operations at 0 to 4”C), drained well and scraped off the dishes with a rubber policeman. The cell suspension (approx. 0.6 ml/l50-mm diam. dish) was homogenized with 3 strokes of a tight-fitting (pestle B) Dounce homogenizer and diluted lo-fold with

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G. KAUFMANN

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buffer B. Portions of 40 ml were layered over 10 ml of 20% (w/v) Ficoll in 10 mM-HEPESNa (pH 7-S), 1 m&@Cl,, 0.5 mM-dithiothreitol, and centrifuged for 10 min at 4000 g in a Sorvall HB-4 rotor. The supernatant was removed, leaving 1 to 2 ml of Ficoll solution above the nuclear pellet. The pellet was then suspended in 45 ml of buffer C and centrifuged for 5 min at 2000 g. Finally, the nuclei were suspended in buffer C to a volume of O-1 to 0.2 ml/l50mm diam. dish of cells used. The nuclei were incubated in the presence of cytosol (DePamphilis & Berg, 1975), nucleotides and salts in reaction mixtures of 1 to 10 ml containing 2 mlvr-rATP, 100 PM each of rUTP, rCTP and rGTP; 1 to 3 PM of radioactively labeled dNTP ([c(-32P]dCTP or [u-~~P]~ATP, 50 to 400 Ci/mmol; or [3H]dTTP, 18 Ci/mmol) and 100 PM of each of the other three dNTPs, 45 mM-HEPES-Na (pH 7+3), 65 mm-KC], 4.6 m&r-MgCl,, 1 mtirEGTAt, 0.3 m&f-dithiothreitol, 0.24 M-ethylene glycol, 5 mm-phosphoenol pyruvate-K, 30 pg pyruvate kinase/ml, 0.6 vol. cytosol and 0.2 vol. nuclei suspension. The reaction was started by addition of nuclei, which were kept at O”C, to the remainder of the mixture at 30°C. The mixture was shaken for 2 min at 30°C. It was then diluted with 10 vol. buffer C at 0°C and centrifuged for 5 min at 2000 g at 0°C. The nuclear pellet was suspended in 1 vol. buffer A and the viral DNA extracted. (d) Isolation

of replicating

SV40

DNA

To the suspended nuclei were added O-14 vol. 6% (w / v i so di urn dodecyl sulfate, 0.1 MTris-HCI (pH 7.8), O-1 M-EDTA (Hirt, 1967) and the mixture was incubated for 5 min at 25’C!, after which 0.28 vol. 5 M-NaCl were added. The mixture was incubated for 4 to 20 h at 0°C and then centrifuged for 2 h at 22,000 g at 0°C. To further purify the viral DNA, the clear supernatant was chromatographed on a Sepharose 6B (Pharmacia) column (2.5 cm x 36 cm) equilibrated with buffer D. The viral DNA emerged immediately after the void volume and was further purified when indicated (see the legend to Fig. 1) by sedimentation through a neutral sucrose gradient followed by banding in a ccsium chloride-ethidium bromide gradient. (e) Fractionation

of single-stranded

DNA

chains

according

to length

The viral DNA, isolated by Sepharose 6B chromatography, was precipitated by adding 3 vol. ethanol. The mixture was centrifuged for 2 h at 114,000 g at -2C. The DNA and heated for 2 min at 50°C. Portions of pellet was dissolved in 98% (v/v) f ormamide 20 ~1 were separated on 12% (w/v) polyacrylamide, 7 M-urea slab gels ( 15 cm x 15 cm x O-15 cm) in 0.1 M-Tris-borate buffer (pH 8*3), 2.5 m&r-EDTA (Peacock & Dingman, 1968). Electrophoresis was carried out at 10 V/ cm at 25°C until the bromphenol blue tracking dye migrated 0.8 to 0.9 of the gel’s length. The gels were calibrated with the following markers : yeast tRNAPhe, yeast tRNAPh” half molecules, RNAase T1 digest of yeast tRNAPhe (Philipsen & Zachau, 1972) and a homologous series of oligo(Ap),. The gel was sliced into 1 mm fractions, and the DNA was electroeluted into dialysis bags containing 0.4 ml of buffer E. The solutions were made up to 0.5 M-NaCl and 30 to 50 pg sonicated DNA/ml (for size change measurements, see below) or yeast tRNA (for label transfer analysis) and the DNA precipitated with 3 vol. ethanol. (f) B’ractionation

of Okazaki

pieces according

to their

position

on the S V40

genome

Okazaki pieces were hybridized to various HindII+III restriction fragments of SV40 DNA. SV40(RI) DNA, isolated by Sepharose 6B chromatography, was dissolved in 99% formamide and the solution diluted with 4 vol. buffer D and again chromatographed on the Sepharose 6B column described above. Long single-stranded DNA chains were excluded from the gel. Fractions containing Okazaki pieces (If,, 0.4 to 0.7) were made up to 50 pg/ml in yeast tRNA and the DNA precipitated with 3 vol. ethanol. The precipitate was dissolved in buffer F ( lo7 ots/min per ml). DNA-Millipore filters containing various HindII+III restriction fragments of SV40 DNA (see below) and a blank filter were soaked in buffer F before being shaken in the solution of Okazaki pieces for 24 h at 37°C. t Abbreviation

used: EGTA,

ethylene

glyool-bis-(@ninoethyl

ether)N,N’-tetraacetic

acid.

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Each filter was then washed with 5 ml of buffer F followed by 20 ml of buffer G. Okazaki pieces were rescued from their hybrids by incubating the filters in 0.5 ml of buffer H for 2 min at 100°C. This extraction was repeated twice, the extracts combined, made up to 0.5 M-NaCl, 50 pg yeast tRNA/ml and the DNA precipitated with 3 vol. ethanol. Hind11 + III restriction fragments were prepared from a digest of 150 pg of SV40 DNA (Danna & Nathans, 1972). Portions containing 50 pg of digested DNA were resolved by electrophoresis in 1.7% (w/v) agarose cylindrical gels (0.8 cm x 20 cm) in buffer E at 4°C for 16 h at 2 V/cm. Gels were stained with O-5 pg ethidium bromide/ml in buffer E and viewed over a long wavelength U.V. lamp. Bands of restriction fragments were excised from the gel, homogenized in 5 ml of saturated KI, and the solution passed through l-ml hydroxylapatite (Bio-Rad, HTP) columns equilibrated in 1 mM-potassium phosphate buffor (pH 6.8). The columns were rinsed with 20 ml of the same buffer before the DNA was eluted with 5 ml of O-5 M-buffer at 60°C. Finally, the DNA was dialyzed against buffer H. DNA-Millipore filters were prepared according to Gillespie (1968) using restriction fragment DNA from 10 pg of SV40 DNA for each 13-mm filter. (g) The 32P-label

transfer

assay

The transfer of 32P from precursor [ a-32P]dNTP to 2’( 3’)rNMPs upon alkaline or RNAase T, incubations of nascent DNA was measured as follows. Samples of 20,000 to 200,000 cts/min of nascent DNA and 10 to 50 pg yeast tRNA were dissolved in 5 to 10 ~1 0.3 MNaOH and incubated for 16 h at 37°C. The solution was then neutralized with 10 vol. 0.03 M-acetic acid, containing 0.005 “/o (w/v) of the tracking dye, xylene cyan01 FF. Alternatively, the sample was dissolved in 10 ~1 of O-1 mg RNAase TJml, a non-specific RNAase (U&da & Egami, 1967), in 0.05 M-sodium acetate (pH 5.7) and incubated for 16 h at 37°C and then diluted lo-fold with 0.005°h xylene cyan01 FF. Before dilution, portions of about O-5 ~1 of either digest were examined by PEI-cellulose thin-layer chromatography with 0.25 M-NH,HC03 to ascertain complete hydrolysis of the carrier RNA. In order to resolve the 32P-labeled 2’(3’)rNMPs from each other and from DNA, the digests were chromatographed on PEI-cellulose thin layers with a solution of 2’(3’) rNMPs (monomixture, see below). The diluted digests were applied to thin layers (10 cm x 20 cm) which were rinsed with water and dried. A triple thick pad of Whatman’s 3MM paper (1 cm x 10 cm) was attached to the upper end of the thin-layer sheet to absorb excess effluent. The thin layers were then developed with 5 ml of monomixture until the dye reached 0.6 to 0.7 of the layer’s height. Monomixture was prepared by dissolving 5 g yeast RNA (Sigma type VI) in 100 ml of 0.27 M-KOH and incubating at 50°C for 3 days before titrating to pH 7.7 with glacial acetic acid. In earlier experiments we used 0.25 M-NH,HCO, as the solvent, which did not separate Cp from Up. The layers were dried, coated with Saran-wrap and autoradiographed. The position of the rNMPs was determined with suitable 32P-labeled markers. To measure the fraction of label transferred, both rNMP and DNA spots were excised from the chromatogram and counted in a toluenebased scintillation fluid. It should be emphasized that in no step was the DNA exposed to acidic conditions. This avoided depurination and subsequent breakdown of DNA (Tiirler, 1971) into low molecular weight compounds which chromatograph like 2’(3’) rNMPs. (h) Assay for oligoribonucleotides

at the 5’ end of nascent DNA

oligoribonucleotide from nascent DNA chains increased The removal of a 5’ terminal their electrophoretic mobility in polyacrylamide gels. The change in DNA mobility was used to detect the presence of oligoribonucleotides and estimate their size. Singlestranded DNA fractions, derived from 1 mm gel slices (see section (e), above) were analyzed. Samples containing 1000 to 2000 cts/min were dissolved in 10 ~1 of 0.01 mg RNAase T,/ml in 0.1 M-sodium acetate, 0.01 M-EDTA and incubated for 4 h at 37°C. Alternatively, the samples were dissolved in 10 ~1 of 0.1 M-NaCH and incubated for 4 h at 60°C and then titrated with 1 ~1 of 1 M-acetic acid. The portions were mixed with 10 ~1 of 50% (v/v) glycerol containing 0.01% each of bromphenol blue and xylene cyan01 FF in 0.1 M-Trisborate (pH 8*3), 2.5 mu-EDTA and analyzed by electrophoresis on 12% or 15% polyacrylamide, 7 M-urea gels, as indicated, along with an untreated sample.

G. KAUFMANN

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ET

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(i) Materials [a-32P]dNTPs were synthesized according to Symons (1974) and further treated with sodium periodate (Neu & Heppel, 1964) to remove traces of ribonucleotides. [3H]dTTP was purchased from New England Nuclear, PI nuclease from P.-L. Biochemicals, RNAase T, from Sigma and PEI-cellulose (Cel 300 PEI, W254) thin-layer sheets from Brinkman Instruments. HindIIfIII endonuclease, purified according to Smith & Wilcox (1970), was a gift from Mr D. Tapper. (j) Buffers

A, 10 mM-HEPES-Na (pH 7-S), 5 mM-KCl, 0.5 mmMg&, 0.5 mm-dithiothreitol; B, 10 mM-HEPES-Na (pH 7+i), 1 mM-Mg&, 0.5 mMdithiothreito1, 0.25 M-sucrose, 0.02% (v/v) Triton X100; C, 10 mM-HEPES-Ns (pH 7.8) 50 mM-KCl, 1 mM-M&l,, D, 10 mm-Tris*HCl (pH 7*5), 1 ~-N&cl, 1 m&rO-5 mnn-dithiothreitol, 0.25 M-SUCPOSR; EDTA; E, 20 mm-TriseHCl (pH 7*5), 40 mM-SOdium acetate, 1 mM-EDTA; F, 0.1 MNaCl, 1 mM-EDTA, 0.5% (w/v) sodium dodecyl sulfate in 50% (v/v) formamide; G, 0.6 M-N&~, 0.06 M-sodium citrate; H, 10 miw-TrisHCl (pH 75), 1 mM-Nacl, 1 mivr-EDTA.

3. Results (a) CopurQkation of RNA-DNA

covalent linkages and replicating 5V40 DNA

The presence of RNA-DNA covalent linkages was demonstrated by the transfer of 32P from a precursor [w~~P]~NTP to 2’(3’)rNMPs upon alkaline incubation of SV40(RI) [32P]DNA. The viral DNA was labeled in isolated nuclei for two minutes at 30°C with [c(-32P]dCTP and purified by (1) isolation of nuclei from the reaction mixture, (2) extraction of viral DNA with sodium dodecyl sulfate and 1 M-NaCl, (3) chromatography on a Sepharose 6B column, (4) sedimentation through a neutral sucrose gradient, and finally (5) centrifugation to equilibrium in cesium chloride/ ethidium bromide. Portions from steps (3) to (5) were assayed for 32P-label transfer and chromatographed as described in Materials and Methods. As shown in Figure 1 and Table 1, a constant fraction of the radioactivity in each digest chromatogram was similarly distributed among the 2’(3’)rNMPs. The levels of these products were TABLE 1 32P-hbel transfer analysis of [cc-~~P]~CTP labeled XV40(RI) during putijiication

Purification step

(3) Sepherose 6B

[=P]DNA (cts/min)

[=P]rNMPs (cts/min)

DNA

3aP transferred to rNMPs (%)

131,300

45

0.015

163,666

274

0.168

(4) Neutral sucrose (hydrolyzed)

181,945

274

0.160

(5) Cesium chloride (hydrolyzed)

92,030

138

0.150

(not hydrolyzed) (3) Sepharose 6B

(hydrolyzed)

DNA and rNMP spots of the ohromstograms shown in Fig. 1 were excised from the thin layer and count,ed in a toluene based scintillation fluid.

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LINKAGES

2’(3’) UP ,;:AP

;‘,:GP

(0)

(b)

(Cl

Cd)

FIQ. 1. Copurification of RNA-DNA linkages and replioat,ing SV40 DNA. SVIO(R1) DNA was pulse-labeled in isolated nuclei with [a-s2P]dCTP (400 Ci/mmol) for 2 min at 30°C. The nuclei were isolated from the mixture and the viral DNA extracted with sodium dodecyl sulfate/NaCl and then further purified by Sepharose 6B gel filtration (Materials and Methods). The DNA was dialyzed against buffer D before sedimenting through a neutral sucrose gradient (5% to 15% linear gradient over a 2-ml 60% sucrose cushion) in buffer D in an SW41 rotor for 14 h at 40,000 revs/min (4°C). The [3aP]DNA fractions were pooled, dialyzed against buffer H and made to 7 ml with buffer H containing CsCl (p = 1.69) and 200 pg ethidium bromide/ ml and centrifuged in a TiSO rotor for 36 h at 45,000 revs/min (16°C). Portions containing SV40 (RI) DNA from the various puriiioation steps were dialyzed against buffer H before precipitating with 3 vol. ethanol and carrier tRNA. The samples were then analyzed for 3aP-label transfer as described in Materials and Methods. Positions of 2’(3’)rNMPs were determined by saP standards (a) Sepharose 6B, unhydrolyzed; (b) Sepharose 6B, hydrolyzed in NaOH; (c) neutral sucrose gradients, hydrolyzed in N&OH; (d) CsCl/ethidium bromide gradient, hydrolyzed in NaOH.

significantly above background (Fig. l(a)). These 32P-labeled mononucleotides were conclusively identified as 2’(3’)rNMPs as described in a separate paper (Anderson et al., 1977). A similar result was obtained with [a-3aP]dATP-labeled SV49(RI) DNA. Alkaline incubation of [32P]DNA also released other products which chromatographed between the origin and Gp. These products originated predominantly from short Okazaki pieces (Pig. 5(a)), and were presumably oligodeoxyribonucleotides. The exposure of 5’-OH termini after alkaline hydrolysis provided independent 36

556

G. KAUFMANN

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evidence for RNA-DNA covalent linkages in SV40(RI) DNA. Nuclei from virusinfected cells were labeled for two minutes at 30°C with [3H]dTTP and the viral DNA isolated, The DNA was digested with Pl nuolease (Wei et al., 1976) before and after incubation under alkaline conditions. The results (Table 2) showed that incubation in alkali exposed 5’-OH term@ which were monitored as thymidine in the Pl nuclease digest. TABLE 2 Exposure

of 5’-OH termini

upon alkaline

hydrolysis

of 8V4O(RI) digest

DNA

and their detection as nucleosides in a PI n&ease Products dTMP (cts/min)

DNA sample

of PI nuclease digestion Thymidine Thymidine (cts/min) (%)

No hydrolysis

64,074

12

0.02

Alkaline

46,970

55

0.12

hydrolysis

Reaction . . . rN-rN-dN-dN 4 Pl PrN+PdN

...B

sequence

OH-

rNP+HO-dN-dN.. 4 p1 HO-dN+PdN

.

SVBO(R1) DNA, labeled with [3H]dTTP, WBS extrscted from the reaction mixture and purified by Sepharose 6B chromatography followed by sedimentation through 8 neutral sucrose gradient 8s described in Materials and Methods and the legend to Fig. 1. Portions of purified SVIO(R1) DNA were dissolved in 0.3 M-NaOH and either titrated immediately with acetic acid to pH 6.8 (not hydrolyzed), or incubated for 16 h at 37°C and then titrated (hydrolyzed). Samples containing 50,000 to 70,000 cts/min were digested at 25°C in O-02 ml of 75 maa-sodium 8cet8te (pH 58), and 0.25 mg Pl nuclease/ml. Samples were applied to cellulose thin-layer sheets (Cel 400 U.V.,,, Brinkman) 8t 30, 60 and 120 min and ohromat,ographed with dTMP and thymidine c8rriers using isobutyric acid/NH,OH/O.l M-EDTA (60: 4O:l) 8s solvent. The chromatograms were sliced and then counted in toluene-based scintillation fluid. DNA ~8s completely converted into dTMP 8nd tmoe smounts of thymidine within 30 min.

(b) Existence of two populations 5’ terminal

oj Okazaki pieces, one with and one without oligoribonucleotides

When pulse-labeled SV40(RI) DNA was denatured and separated by gel electrophoresis (Fig. 2) about 50% of the label incorporated into DNA was found in chains shorter than 200 nucleotides (Okazaki pieces). These chains had a number average length of less than 100 nucleotides (Fig. 2). Upon alkaline incubation, these chains would have transferred at least 1% of their radioactivity (or O6o/o of the total in SV40(RI) DNA) to 2’(3’)rNMPs had all of them carried a a2P-labeled 5’ terminal deoxynucleotide covalently linked to RNA. However, the 32P-label transfer values obtained with each of the four precursor [cr-32P]dNTPs (Table 1; Anderson et al., 1977) fell short of this expectation. This discrepancy was due to the existence of two populations of Okazaki pieces, one with and one without RNA linked to DNA. Thus, when Okazaki pieces of defined length were isolated by gel filtration and then analyzed by centrifugation to equilibrium in potassium iodide gradients, the polynucleotide chains were distributed between a peak at the density of single-stranded

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LINKaGES

I

Distance from origin (nm) L

,

.

175

120

00

53

36

24

Chain length (nucleotidesl

FIQ. 2. Electrophoresis of denatured SV40(RI) DNA on a polyacrylamide/urea gel. SV40(RI) DNA labeled with [a-32P]dCTP was purified and then denatured and fractionated by electrophoresis on a 12% polyacrylamide, 7 x-urea gel as described in Materials and Methods. The gel was sliced into 1 mm portions which were counted by the cerenkov procedure. The i?zsert shows the relative number of chains ver8u8 the chain length in nucleotides. The Okazaki range (10 to 120 mm) was divided into lo-mm sections and the radioactivity from each section integrated. The ratio (R) was taken as the relative number of chains at a defined chain length.

R=

$J cts/min +?J xi - xi’

where z” and q,, are the delimiting lengths of the section. The chain number vers2cs chain length pattern varied somewhat from experiment to experiment. In particular, we noted shorter than usual chain populations when 1 prd-dCTP was used.

DNA and a broad shoulder at a density commensurate with an RNA content of about 10% (Fig. 3). Since these data could have resulted from an artefactual aggregation of RNA with DNA in the high salt gradients (Probst et al., 1974; Reichard et al., 1974) the presence of two populations of Okazaki pieces was further demonstrated in an independent experiment. If RNA-DNA covalent linkages in nascent DNA are considered to originate from 5’ terminal oligoribonucleotides (Reichard et al., 1974), then their removal should result in a small discernible shortening of the DNA chains. Indeed, after Okazaki pieces were isolated from 1 mm polyacrylamide gel slices (Fig. 2) and treated with either RNAase T, or potassium hydroxide, a discrete subfraction, comprising about one-third to one-half of the DNA chains, migrated as if shortened by about seven nucleotides (Fig. 4). The calculated difference in size was based on the assumption that both populations had 5’-OH termini. It is likely, however, that the excised oligoribonucleotide carried a 5’ mono- or polyphosphate group (Reichard et al., 1974). If this were the case, then the size difference would be greater than seven nucleotides and possibly as large as ten nucleotides. Since the new, faster moving band had the same narrow chain length range as the original band, we conclude that the 5’ oligoribonucleotide had a unique length. We will refer to this 5’ terminal oligoribonucleotide as an RNA primer.

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AL.

Fraction number

FIU. 3. Distribution of Okazaki pieces in potassium iodide equilibrium density gradients. SV40(RI) DNA, labeled with [a-32P]ATP (-O-O-) and purified by sedimentation through a neutral sucrose gradient, was denatured and chromatographed on a Sepharose 6B column as described in Materials and Methods. Portions of 20,000 cts/min from fractions containing Okszaki pieces of defined chain length were made to 7 ml in 0.01 M-NaHSO,, 0.01 M-potassium phosphate buffer (pH 6.8) and KI to p = 1.54 (DeKloett & Andrean, 1971). Denatured SV40(11) [3H]DNA (-a-@-) was added as a marker. The solution was centrifuged in a Ti50 Beckman rotor for 48 h at 46,000 revs/min (0°C). saP in each fraction was counted by the cerenkov procedure. To determine the position of the eH label, the fractions were mixed with 0.5 ml of a 0.2% (w/v) hydroxylapatite suspension in 0.01 M-potassium phosphate (pH 6.8), applied to Whatman’s GF/C filter circles, washed with water and dried and counted in toluene-based scintillation fluid. The density across the gradient was determined by weighing 200-~1 samples in constriction pipets calibrated with water. The polynucleotide chains in the shoulder were estimated to be 10 mg/ml denser than single-stranded DNA. This corresponds to about 10% RNA content (DeKloett & Andrean, 1971). (a) Chains of 75 to 150 nucleotides; (b) chains of 50 to 120 nuoleotides as determined by gel electrophoresis. ss, single-stranded; ds, double-stranded.

(c) DNA chaiv~, with and without RNA primers, exist among all sizes of Okazaki pieces The question arose as to whether the two populations of Okazaki pieces were similarly distributed throughout the range of nascent DNA chain lengths or whether RNA primers were predominantly linked to short DNA chains because they were excised during DNA chain elongation. To distinguish between these possibilities, 32P-label transfer analysis and 5’ terminal oligoribonucleotide determinations were done on DNA chains of various lengths. SV40(RI) DNA, pulse-labeled with [a-32P]dCTP, was denatured and the [32P]DNA fractionated according to length by either Sepharose 6B chromatography, to recover long DNA chains, or by polyacrylamide/urea gel electrophoresis to fractionate Okazaki pieces into defined size classes. Figure 5 and Table 3 present results of 32P-label transfer analysis on different lengths of nascent DNA using RNAase T,. The shortest chains (21 nuoleotides in experiment 1; I3 nuoleotides in experiment 2) contained about 0.6 rN-32P-dC linkages per chain while longer chains, up to 150

559

b)

10

100

90

80 Distance

from crigin

110

(mm)

FIG. 4. Removal of RNA primers from Okazaki pieces upon RNAase Ta or alkaline digestion.

(a) Single-stranded DNA fractions of the chain lengths indicated were obtained ae described in the legend to Fig. 2 and Materi& and Methods. Portions containing 600 to 2000 cts/min were digested rend then electrophoresed on & 16% polyacrylemide, 7 M-urea gel. C is undigested chains, T!2 &&ains inea?x&ed wit% KmAaae Fa, OH is chains incubated with alkali. Chain lengths were 54, a; 33, b; 21, o; 63, d nuoleotides. The streak of small polynuoleotides seen in fractions a, b, and o did not result from the digestion procedures, because the same streak w&s also observed in undigested controls. Furthermore, it did not occur in other preparations, e.g.d. (b) Miorodensitometer tracing of chains 33 nucleotides long before (b- C) ( ) ma after (b- T2) (-----------) treatment with RNA&se T,. had a ratio of O-2 to O-3. DNA chains of about 400 nucleotides oontained RNA-DNA covalent linkages at a ratio calculated to be 0.3 per chain (see the legend to Table 3). 3aP-label transfer analysis yielded identical results whether done with either RNA&se T, or potassium hydroxide (Table 4). Duplicate assays on the same DNA sample agreed within f5% (Table 4), although the DNA chain lengths containing higher frequencies of rN-32P-dC linkages varied among independent experiments (Table 3). nucleotides,

G. KAUFMANN

A60

El’

AI,.

CP UP -

-

AP -

(01

lb)

(cl

(d)

(el

CP,UP

-

AP

-

GP

(f)

FIG 5. 32P-label transfer analysis on DNA-chains of various lengths. Fractions of [a-3ZP]dCTP-labeled Okazaki pieces of defined length were electroeluted from IO-mm sections of a polyacrylamide gel (see Fig. 2). Longer chains were obtained by Sepharose 6B chromatography (Materials and Methods). ssPJabe1 transfer analysis was carried out with RNAase Ts and chromatographed as described in Materials and Methods. Median chain length: (a) 21; (b) 84; (c) 104; (d) 128. (e) Long chains not hydrolyzed; (f) long chains, above 400 nucleotides.

A parallel analysis of the occurrence of 5’ terminal oligoribonucleotides in Okazaki pieces of various lengths revealed that the length of the RNA portion removed by either RNA&se T, or potassium hydroxide remained constant at about seven nuclep, tides (Table 5). The two discrete populations of Okazaki pieces, one with, and ‘one without an oligoribonucleotide, occurred in a similar ratio throughout the range of DNA chain lengths examined (Table 6). These data show that the rN-3aP-dN linkages present could only have come from the 5’ termini of DNA chains, and must have resulted from covalent linkage to the uniquely sized oligoribonucleotides. Thus, about one-third to one-half of all the Okazaki pieces carried an RNA primer. (d) Uniform distribution of RNA-DNA linkages around the replicating genome To determine whether RNA-DNA covalent linkages emanate from specific sites on the 8V40 genome such as the origin of replication, 32P-label transfer analysis was performed on [a-32P]dCTP-labeled Okazaki pieces recovered after hybridization

SIMIAN

VIRUS

40 RNA-DNA TABLE

LINKAGES

561

3

The ratio of RNA-DNA covalent linkages per nascent DNA chain in DNA jrachns of various chain lengths Experiment

no.

-wft

2P19 92-76 115-92 141-115 >400

1

21 84 104 128 uw

16-10 23-19 37-30 49-61

2

Fraction of aaP transferred to rNMPs

DNA chain length Median

8

13 21 34 65

Number rN-=P-dN linkages per chaint

0.0280 0.0037 0.0027 0.0024 0.0028

0.59 0.31 0.28 0.31 0.28§

0.0436 0.0080 0.0069 0.0047

0.57 0.17 0.23 0.26

DNA and rNMP spots were cut from PEI-cellulose thin layers (Fig. 6) and counted in a toluenebased scintillation fluid. t Five residues were subtracted from the actual delimiting chain lengths (Fig. 2), assuming that about half of the Okazaki chains carried a lo-residue long RNA primer (see Discussion). $ The product of the fraction of saP in DNA transferred to rNMPs and the median DNA chain length. The incidence of dC in RNA-DNA linkages was assumed to reflect its incidence in DNA. f The labeled portion of long chains was calculated to be about 100 nucleotides. This was based on our observation that, synthesis of a 200-nucleotide Okazaki piece required about 2 mm, and on the assumption that DNA synthesis was completely discontinuous. A semidiscontinuous model (Francke & Hunter, 1974) or intermediate oases yielded lower estimates for the length of the labeled portion in long chains, and therefore higher estimates of the ratio of RNA-DNA linkages per chain. TABLE

4

Comparison of 32P-label transjer analysis using either RNAase T, or alkaline incubation Inoubation [=P]DNA

of

RNAase

Tz

DNA (aaP cts/min)

rNMPs (32P cts/min)

Fraction transferred

of 3aP to rNMPs

31,800

76

0.0024

Alkaline

31,990

76

0.0023

None

36,800

7

0*0002

32P-label transfer analysis was done as described in Materials and Methods with pieces with a median DNA chain length of 128 nucleotides labeled with [a-3sP]dCTP.

Okazaki

to various HindII+III restriction fragments of SV40 DNA. Nascent DNA recovered from one restriction fragment did not anneal to other fragments. It was found that Okazaki pieces originating from different segments of the genome contributed similar levels of 2’(3’)rNMPS with no significant differences in the pattern with which the transferred label was distributed among the four 2’(3’)rNMPs (Pig. 6; Table 7). Similar results were obtained with [cr-32P]dATP-labeled Okazaki pieces. RNA primers originated from all regions of replicating viral DNA.

ET

G. KAUFMANN

562

AL.

TABLET

The size of RNA primers on Okazaki pieces of various lelagths Original

Apparent size change after RNAase Ts or KOH incubation (nucleotides)

DNA chain length (nucleotides)

-7 -8

16 21 26 33 42 56 66 80 93 98 117

-8 -8 -7 -8 -6 -7 -6 -7 -7

The apparent change in the length of Okazaki pieces upon RNAase T, was calculated from the center-to-center distance between the undigested moving band appearing after digestion ss shown in Fig. 4(a). Chains of 66 were analyzed in 12% polyacrylamide/urea gels; chains of 66 nucleotides 15% gels.

TABLE

or alkaline incubation control and the faster nucleotides and above and less were done on

6

The fraction of Okazaki pieces carrying RNA primers Original DNA chain length (-RNAase T,) (nucleotides)

Fraction of DNA chains with RNA primers (+RNAase T,) (%)

21 36 54 63 80,98,117

35 42 48 w (40-60) $

The amount of radioactivity in nascent DNA chains before and after incubation with RNA&se T, was determined using a microdensitometer to trace the autoradiogram shown in Fig. 4(s) (see Fig. 4(b)). In converting the observed DNA chain weight ratio into a number ratio, the faster moving chains were considered to contain 11 labeled phosphates fewer than the slower chains. This allows for the loss of a IO-nucleotide RNA primer and the 6’ terminal phosphate of the DNA chains. t This result wss teken from an independent experiment. $ The fraction of Okazaki pieces carrying RNA primers in chains longer than 80 nucleotides was visually estimated to be O-4 to 0.6. These bands were too close for the densitometer to adequately resolve them. However, this diEiculty did not effect the center-to-center distance measurements used in Table 6.

4. Discussion The results presented in this paper demonstrate that the RNA-DNA covalent linkages which copurify with replicating SV40 DNA can be accounted for by the presence of oligoribonucleotides covalently linked to the 5’ termini of a portion of the nascent DNA chains, This con&ms previous reports about the involvement of RNA

SIMIAN

VIRUS

40 RNA-DNA

563

LINKAGES

2’(3’lCp273’)

up -

2’ AP 3’Ap-

Z’(3’)Gp

-

(F,G)

(E)

(C,O)

(A)

FIa. 6. 3ZP-18bel transfer analysis around replicating SV40 DNA. Okazaki pieces, lebeled with [m-3ZP]dCTP and recovered from hybrids with the indicated HindII+III restriction fragments of SV40 DNA, were incubated with KOH and analyzed for transfer of saP to rNMPs as described in Mat.erials and Methods.

in discontinuous DNA synthesis in various eukaryotic organisms (Magnusson et al., 1973; Francke & Hunter, 1974; Tseng & Goulian, 1975; Waqar & Huberman, 1975a,b). The size of the oligoribonucleotide removed from one population of SV40 Okazaki pieces upon incubation with RNAase T, or potassium hydroxide was similar to that of the oligoribonucleotide found at the 5’ end of nascent polyoma DNA chains (Reichard et al., 1974). SV40 RNA primers are of a unique size of about seven residues, since the length range of DNA chains after their RNA primers were removed was no broader than that of the original chains (Fig. 4). 32P-label transfer analysis of Okazaki pieces containing 20 to 150 nucleotides detected rN-32P-dN linkages in about one-third of the DNA chains (Table 3). In contrast, about one-half of these chains carried a terminal oligoribonuoleotide (Fig. 4; Table 6). These oligoribonucleotides must be covalently linked to the 5’ ends of

O-226 o-205 0.086 O-146

Fractional length-t

Term’Llus (DNA replication)

0.33 o-43 0.38 0.34

ZrNMPs

LIindII+III

O-08 0*08 0.08 0.07 restriction

0.11 o-20 0.14 0.12

AP 0.08 0.10 o-09 0.06

GP

A (DNA

Origin replication)

0.18 0.22 0.07 0.10

rC-dC 0.13 o-14 0.06 0.11

O-24 04i6 0.12 0.17

Molar freotion$ rU-dC rA-dC

O-18 0.27 0.07 0.08

rG-dC

I t c I = I E lKIFl

enzyme map of SV40 DNAt

0.06 O-06 0.07 O-08

o/0 =?? transferred to : CP UP

B III HI

16,661 18,611 16,642 18,342

[32P]DNA (cts/min)

DNA and rNMP spots were excised from the thin-layer chromstogram (Fig. 6) and counted in a toluene-based scintillrttion fluid. t The fractional length of HindIIfIII DNB fragments and the HindII+III restriction enzyme map of SV40 DNA w&s taken from the data of Dana, et al. (1973). $ The molar fraction of rN-3aP-dC junctions in a given restriction fragment assumes that Okazaki pieces originated from both arms of the replication fork and were, on the average, half their mature size. The number of dCMP residues in each restriction fragment was calculated acoording to the nucleotide composition data given by Denns t Nathrtns (1972). Molar fraction of rN-3ZP-dC = (number of dCMP residues in the fragment) (fraction of 32P transferred t,o rNMPs)

RI site

i+lGI + f

A C+D E F+G

HindI + III DNA fragments

TABLET The distributiolz of rN-32P-dC linkages around S V40( RI) DNA

SIMIAN

VIRUS

40 RNA-DNA

LINKAGES

565

Okazaki pieces, since hydrolysis of rN-P-dN linkages did not produce a broad distribution of DNA chain lengths. This suggests that not all of the RNA-DNA junctions were labeled during the pulse in vitro ; some of the nascent DNA chains had already incorporated one or more deoxyribonucleotide. However, about one-half of the very short DNA chains (20 nucleotides or less) contained labeled RNA-DNA junctions (Table 3). Since there appeared to be an excess of these chains (Fig. 2, insert), some of them may represent initiation of Okazaki pieces that were subsequently aborted. The presence of two populations of Okazaki pieces, one with and one without RNA primers, could not be explained by a gradual excision of the primer during DNA chain elongation, although rapid excision of a putative RNA primer from one population cannot be ruled out. RNA primers remained linked intact to Okazaki pieces throughout a wide range of chain lengths. Furthermore, at least the 3’ terminal nucleotide of the primer must have been carried onto long chains as shown by saPlabel transfer analysis (Table 3). Thus, complete excision of the RNA primer appears to occur only after the Okazaki piece has been joined at its 3’ end to a long chain, as expected to be the case on the retrograde arm of the replication fork (Gefter, 1975). Several explanations for the existence of two populations can be imagined. We are currently examining the possibility that the two populations arise from different arms of the replication fork. Alternatively, some Okazaki pieces may result from artificial or natural breaks inflicted on the primary products of synthesis (Tye et al., 1977). However, if mature Okazaki pieces are variable in length rather than homogeneous, then those without RNA may simply represent Okazaki pieces whose primers have been removed but which have not yet been joined to growing daughter strands. Finally, primer synthesis may utilize either rNTPs or d.NTPs depending on their relative concentrations ; Okazaki pieces without an RNA primer may actually contain a DNA primer. Analysis of Okazaki pieces recovered from hybrids with various H&dII+III restriction fragments of SV40 DNA showed that rN-3aP-dC (Table 7) and rN-3aP-dA sequences (not shown) were uniformly distributed around SV40(RI) DNA. Therefore, most, if not all of the RNA primers serve to initiate Okazaki pieces rather than DNA replication at the origin of the genome. Each of the regions of the SV46 genome examined could accommodate only 4 to 12 Okazaki pieces, of which only two to six chains would carry RNA primers. Were the initiation signals for these primers determined by specific nucleotide sequences, each fragment would accommodate a small subset of the 16 possible rN-P-dN sequences, However, we found that the four rN-P-dC sequences alone were similarly represented in all segments of the genome, each at small fractional molar ratios. Hence, synthesis of Okazaki pieces must begin at different points on different replicating genomes. Since the RNA-DNA junctions are synthesized independent of DNA template sequence (see also Anderson et al., 1977) and the length of RNA primers is unique, then the 5’ termini of RNA primers must also occur independent of DNA template sequence. We propose that the signal for initiation of Okazaki pieces is provided by the chromatin subunit structure, the nucleosome. Nucleosomes contain about 206 nucleotide pairs of DNA (Kornberg, R. D., 1974; Noll, 1974; Hewish & Bourgoyne, 1973) similar to the maximal length of Okazaki pieces (Fig. 2; Kornberg, R. D., 1974; Fareed et al., 1972; Magnusson et al., 1973; Hunter t Francke, 1974; Tseng & Goulian,

566

G. KAUFMANN

ET

AL.

1975). Neither the nucleosomes (Polisky & McCarthy, 1975; Cremisi et aE., 1976) nor the initiation signals for Okazaki pieces (Table 7) are uniquely positioned on the genome. However, the nucleosomes are regularly spaced (Cremisi et al., 1976) and could provide regular signals for synthesizing RNA primers that are independent of DNA sequence. In order to form a stable RNA-DNA hybrid, RNA polymerases require a single-stranded DNA template (Chamberlin & Berg, 1964; Champoux $ McConaughy, 1975). Since the DNA in each nucleosome is deficient in about 1.5 to 2 double helical turns (Germond et al., 1975; Shure & Vinograd, 1976), each nucleosome provides a potential template, 10 to 20 nucleotides long. This deficiency is presumably spread uniformly along the nucleosomal DNA (Sobel et al., 1976; Weintraub et al., 1976), but may become clustered into one or more template regions on dissociation of the nucleosome during DNA replication.

Cytosol was prepared from CV-1 cells grown by the Massachusetts Institute of Technology Cell Culture Center. This research was supported by National Institutes of Health grant CA 155’79-03. One of us (G. K.) was supported in part by a fellowship from the European Molecular Biology Organization, another (S. A.) was supported by National Service Award CA 09031. The other author (M. L. D.) is an Established Investigator of the American Heart Association. REFERENCES Anderson, S., Kaufmann, G. & DePamphilis, M. L. (1977). Biochemistry, in the press. Brutlag, D., Scheckman, R. & Kornberg, A. (1972). Proc. Nat. Acad. Sci., U.S.A. 68, 28262829. Chamberlin, M. & Berg, P. (1964). J. Mol. Biol. 8, 227-313. Champoux, J. J. & McConaughy, B. L. (1975).. Biochemistry, 14, 307-316. Cremisi, C., Franc0 Pignatti, P., Croissant, 0. & Yaniv, M. (1976). J. Viral. 17, 204,211. Danna, K. J. & Nathans, D. (1972). Proc. Nat. Acad. Sci., U.S.A. 68, 3097-3100. Danna, K. J., Sack, G. H. & Nathans, D. (1973). J. Mol. BioZ. 78, 363-376. DeKloett, S. R. & Andrean, B. A. G. (1971). Biochim. Biophye. Acta, 247, 519-527. DePamphilis, M. L. & Berg, P. (1975). J. BioZ. Chem. 250, 4348-4354. DePamphilis, M. L., Beard, P. & Berg, P. (1975). J. BioZ. Chem. 250, 4340-4347. Edenberg, H. J. & Huberman, J. A. (1975). Annu. Rev. Geaet. 9, 245-284. Elliasson, R., Martin, R. & Reichard, P. (1974). Biochem. Biophye. Res. Commun. 59, 307-313. Fareed, G. C., Garon, C. F. & Salzman, N. P. (1972). J. Viral. 10, 484-491. Francke, B. & Hunter, T. (1974). J. Mol. BioZ. 83, 99-121 Gefter, M. (1975). Annu. Rev. Biochem. 44, 45-78. Germond, J. E., Hirt, B., Oudet, P., Gross-Bellard, M. & Chambon, P. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 1843-1847. Gillespie, D. (1968). Methods Enzymol. 12B, 641-646. Girardi, A. J. (1965). Proc. Nat. Acad. Sci., U.S.A. 54, 445-450. Helinski, D. (1976). Fed. Proc. Fed. Amer. Sot. Exp. BioZ. 35, 202&2030. Hewish, D. R. & Bourgoyne, L. A. (1973). Biochem. Biophys. Res. Commun. 52, 504-510. Hirose, S., Okazaki, R. & Tomanoi, F. (1973). J. Mol. BioZ. 77, 501-517. Hirt, B. (1967). J. Mol. BioZ. 26, 365-369. Hunter, T. & Francke, B. (1974). J. Mol. Biol. 83, 123-130. Kornberg, A. (1974). DNA Synthesis, Freeman, San Francisco. Kornberg, R. D. (1974). Science, 184, 868-871. Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. & Okazaki, R. (1975). J. Mol. Biol. 96,653-664. Magnusson, G., Pigiet, V., Winnacker, E. L., Abrams, R. & Reichard, P. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 412-415.

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Rowen, L. S. & Bouche, J. P. (1976). Fed. Proc. Fed. Amer. Sot. Ezp. BioZ. 35, 1418. W. P. (1973). Biochem. Biophys. Res. Commun. 53, 818823. Sadoff, R. B. 8s Chewers, Shure,

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Smith, H. 0. t Wilcox, K. (1970). J. 1MoZ.BioZ. 51, 379-391. Sobel, H. M., Tsai, C., Gilbert, S. G., Jain, S. C. & Sakore, T. D. (1976). Proc. Nut. Acad. Sk.,

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Sugino, A. & Okazaki, R. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 88-92. Sugino, A., Hirose, S. & Okazaki, R. (1972). Proc. Nut. Acad. SC&, U.S.A. 69, 1863-1867. Symons, R. H. (1974). Metbds Enzymol. 29, 102-115. Tseng, B. Y. & Goulian, M. (1975). J. Mol. BioZ. 99, 339-346. Tiirler, H. (1971). In Procedures in. Nucleic Acid Research (Cantoni, G. L. & Davis, D. R., eds), vol. 2, pp. 686-702, Harper and Row, New York. Tye, B. K., Nyman, P. O., Lehman, I. R., Hochhauser, S. & Weiss, B. (1977). Proc. Nat. Acad.,%.,

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Uchida, T. & Egami, F. (1967). Methods Enzymol. 12A, 239-247. Waqar, A. & Huberman, J. A. (1975a). Biochim. Biophys. Acta, 383, 410-420. Waqar, A. & Huberman, J. A. (19753). CeZZ,6, 551-557. Wei, C. M., Gershowitz, A. & Moss, B. (1975). Nature (London), 252, 251. Weintraub, H., Worcel, A. & Alberta, B. (1976). Cell, 9, 409-417. Wells, R. D., Fliigel, R. M., Eason, J. E., Schendel, P. I. & Sweet, R. W. (1972). Biochemistry,

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