Elongation of primed DNA templates by eukaryotic DNA polymerases

2 downloads 0 Views 1MB Size Report
Jul 14, 1980 - In addition, aphidicolin, a selective in- hibitor of Pol a in vitro (7, 9, 10), inhibits DNA synthesis in. HeLa cells (11), sea urchin embryos (12), and ...
Proc. Natl. Acad. Sci. USA

Vol. 77, No. 10, pp. 5827-5831, October 1980 Biochemistry

Elongation of primed DNA templates by eukaryotic DNA polymerases -

(replication/adenoviral DNA)

JOH-E IKEDA*, MATHEW LONGIARUt, MARSHALL S. HORWITZt, AND JERARD HURWITZ* *Department of Developmental Biology and Cancer, tDepartment of Microbiology and Immunology, Division of Biological Sciences, Albert Einstein College

of Medicine, Bronx, New York 10461

Contributed by Jerard Hurwitz, July 14,1980

ABSTRACT The combined action of DNA polymerase a and DNA polymerase P leads to the synthesis of full-length linear DNA strands with #X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase a catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase P elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase a catalyzes only limited deoxynucleotide addition whereas DNA polymerase P alone elongates DNA primed templates to full length. DNA polymerase ,can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA)oligo(dT) as the primer template.

Fwas

Three DNA polymerases (Pols)-a, fl, and y-have been isolated from eukaryotic cells (1). Circumstantial evidence suggests that Pol a is involved in DNA replication. Synthesis of HeLa cell DNA by cell lysates is resistant to 2',3'-dideoxythymidine triphosphate (ddTTP), which does not affect Pol a but inhibits Pol 1 and Pol y (2-8). In addition, aphidicolin, a selective inhibitor of Pol a in vitro (7, 9, 10), inhibits DNA synthesis in HeLa cells (11), sea urchin embryos (12), and extracts of regenerating rat liver (13). Pol a, the major Pol associated with the replicating simian virus 40 chromosome, participates in the in vitro DNA synthesis by the simian virus 40 replication complex (2-4). Pol 1 has been considered to be a repair enzyme (5, 14) although Eichler et al. (15) found that Pol 1 was capable of extending D loops of replicating mitochondrial DNA. Pol y is the only polymerase present in mitochondria (16-18) and it has been assumed that this enzyme is important in replication of mitochondrial DNA. To date, none of the known Pols of prokaryotic or eukaryotic origin initiate DNA synthesis de novo. All require a primer which may be an oligoribonucleotide or an oligodeoxyribonucleotide having a free 3'-OH end (19). Evidence that RNA serves to prime DNA synthesis has been reported for Escherichia coil, bacteriophages, simian virus 40, and HeLa cells (20). As part of a study of eukaryotic DNA synthesis, we have examined the role of eukaryotic Pols in the elongation of RNAprimed kX174 DNA and of elongation of minute virus of mice (MVM) DNA (a DNA-primed template) and in an in vitro adenovirus (Ad) DNA replicating system (21, 22). Our results suggest that, with limiting amounts of primer ends, Pol a elongates RNA primers to a limited extent whereas Pol 1 and Pol y cannot utilize RNA primers. In the presence of DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5827

primers, Pol /3 catalyzes extensive elongation but Pol a and Pol ,y are inactive. The combination of Pol a and Pol 1 leads to synythesis of 4X174 RF II (a double-stranded circular structure in which one strand is not covalently closed) with RNA-primed qX174 DNA. In addition, Pol 13 preparations stimulate the synthesis of Ad DNA in an in vitro replication system which initiates and elongates Ad DNA to full length.

MATERIALS AND METHODS Synthesis of OX DNA-RNA Hybrid. DNA from phage /X174 was isolated (23) and OX DNA-RNA hybrids were prepared as described (24) except that the incubation with RNA polymerase was increased to 20 min at 30°C. The RNA present in the hybrid form was 600-700 nucleotides long and was analyzed as described by McMaster and Carmichael (25). Elongation reactions catalyzed by E. coli Pol III, dnaZ protein, and DNA elongation factors I and III (26) indicated that 25% of the input OX DNA-RNA hybrid yielded RF II products. More than 80% of the DNA chains formed by the E. coli system with the RNA primer yielded RF II molecule about 4700 nucleotides long, corresponding to a full-length linear strand. RF II formation was abolished when hybrids were treated with RNase A plus RNase T1. The parvovirus MVM DNA was a generous gift from D. C. Ward (Yale University). ['4C]Thymidine-labeled Ad DNA covalently bound to a 55,000-dalton protein at the 5'-end of each strand (Ad DNA-pro complex) was purified from Ad virions as described (22). Nicked salmon sperm DNA was prepared as described (9). HeLa Cell Pols. Pols a, 1, and y were purified from HeLa cell nuclei as described (9). One unit of DNA polymerase was defined as the amount which incorporated 1 nmol of dTMP in 20 min at 30°C. These preparations, up to 1 ,g (maximum tested), contained no nuclease or ATPase activities (with or without DNA). Conditions for in Vitro DNA Elongation. Reaction mixtures (50,ul) containing 20 mM Tris-HCl (pH 7.5 at 30°C), 3 mM MgCl2, 2 mM dithiothreitol, 0.1 mM NAD+, 1 mM spermidine, dATP, dGTP, and dCTP at 80 ,uM each, 40 uM [3H]dTTP (1500 cpm/pmol) or [a-32P]dTTP (6000-20,000 cpm/pmol), 2 mM ATP, 0.2 mg of bovine serum albumin per ml, 25 mM NaCl, OX DNA-RNA hybrid (57 ng) or MVM DNA (46 ng), and Pol a (0.02 unit) or Pol 1 (0.026 unit) or Pol y (0.02 unit) as indicated were incubated for 60 min, and dTMP inAbbreviations: RF I, superhelical, double-stranded, closed circular DNA; RF II, duplex structure in which one strand is not covalently closed; RF III, double-stranded, full-length linear 4X174 DNA; Ad, adenovirus; Ad DNA-pro, adenovirus DNA covalently bound to a 55,000-dalton protein at the 5'-end of each strand; MVM, minute virus of mice; MalNEt, N-ethylmaleimide; ddTTP, 2',3'-dideoxythymidine 5'-triphosphate; Pol, DNA polymerase.

5828

Biochemistry:

Ikeda et al.

corporation into acid-insoluble material was measured. For analysis of products by agarose gel electrophoresis, reaction mixtures were adjusted to 10 mM EDTA and 0.1% sodium dodecyl sulfate. Agarose Gel Electrophoresis of DNA Product. Reaction mixtures treated as described above were subsequently incubated with proteinase K (0.2 mg/ml) at 37°C for 60 min, and then NaCl (0.1 M) and OX DNA (1 gg) were added. The [132PJdTMP-labeled products were chromatographed on Bio-Gel A-5m column (100-200 mesh, 4 ml) and precipitated with ethanol. For native agarose gel electrophoresis, samples were dissolved in 10 mM Tris.HC1, pH 7/5 mM EDTA/10 mM NaCI (TES buffer) containing 0.4% Sarkosyl, 0.02% xylene cyanol, 0.02% bromphenol blue, and 10% (vol/vol) glycerol, applied to a 1.2% agarose (50 mM Tris base/40 mM sodium acetate, pH 7.9/1 mM EDTA) slab gel (3 mm thick, 15 cm wide, 12 cm long), and electrophoresed at room temperature for 4 hr at 50 V. For alkaline agarose gel electrophoresis, alcohol-precipitated samples were dissolved in 0.3 M NaOH/5 mM EDTA/0.4% Sarkosyl/0.02% xylene cyanol/0.02% bromphenol blue/10% (vol/vol) glycerol, applied to a 0.8% agarose/30 mM NaOH/1 mM EDTA slab gel (3 mm X 15 cm X 12 cm), and electrophoresed at room temperature for 4 hr at 50 V. RESULTS DNA Synthesis on Primed DNA Templates. Two DNA templates containing different primers were examined in detail one containing RNA and the other containing a DNA hairpin as primer. Both templates were approximately the same length (-4400 nucleotides long). Both templates were scored for available 3'-hydroxyl ends with the E. coli Pol III elongation system. In subsequent experiments, the number of 3'-hydroxyl ends of primed templates used per assay was held constant. Synthetic templates as well as nicked salmon sperm DNA were Table 1. Utilization of different primed templates by Pol dTMP incorporated, pmol With OX With DNA-RNA Pol added MVM DNA hybrid 0.59

0.18 7.48 0.05 0.05 ly 3.92 7.57 a + ,8 0.51 0.20 a + 'Y 3.97 7.42 a + 0 + -Y a* ND 0.05 1.88 0.06 ND a + ,B* 0.77 ND 0.72 a* +13 The complete system (0.05 ml), containing OX DNA-RNA hybrid (57 ng) or MVM DNA (46 ng) or poly(dT)-oligo(rA) (4 Mg) was incubated at 30°C for 60 min. Reaction mixtures containing nicked salmon sperm DNA (10 ,g) or poly(rA).oligo(dT) (4MLg) were as described (9) except that the final concentration of NaCl was 25-30 mM and mixtures were incubated at 300C for 20 min with 90, 58, or 50 ng of Pol or -y, respectively. Treatment of polymerases with 10 mM Na, ethylmaleimide (MalNEt) prior to addition to reaction mixtures was carried out as described (23). Reactions with nicked salmon sperm DNA and Pol a resulted in 21.8 pmol dTMP incorporated; this value was reduced to 0.08 pmol after MaINEt treatment. Incorporation of dAMP with poly(dT)-oligo(rA) was 5.7 and 19.1 with Pol a and Pol respectively-, the combination of Pol a plus Pol yielded 23.5 pmoL Incorporation of dTMP with poly(rA)-oligo(dT) with Pol ,B and Pol ,y was 13.1 and 18.6 pmol, respectively. MalNEt treatment yielded 15.4 and 0.3 pmol with Pol and Pol 'y, respectively. ND, not determined. * Pol treated with MalNEt. a

0.92

13,

13,

Proc. Natl. Acad. Sci. USA 77 (1980) 1

ITi 11 RIE 111 -

3 9

2

3

t

1

ii

-

9

1(

S

O\ D)NARHNA-

FIG. 1. Structure of DNA synthesized with OX DNA-RNA hybrid by Pol a and Pol P3. OX DNA-RNA hybrid (57 ng) was incubated for 60 min at 300C with Pol a (90 ng) or Pol 13(58 ng) or both. Products labeled with [a-32P]dTTP were subjected to neutral agarose gel electrophoresis. Radioautography was performed by exposing gels at -70°C for 20 hr (Kodak XR-5 film). Lanes: 1, products synthesized with Pol a plus Pol 13; 2, OX DNA-RNA hybrids preincubated with RNases A (2 Ag/ml) plus T1 (10 units/ml) at 37°C for 30 min followed by incubation with Pol a and Pol ,B; 3, after 10 min of incubation, RNases A (2 Mg/ml) and T1 (10 units/ml) were added to reactions as in lane 1; 4, products were synthesized without ATP under conditions described for lane 1; 5, reactions as in lane 1 without MgCl2; 6, reactions as in lane 1, in the presence of aphidicolin (10 gM); 7, products synthesized with Pol a alone; 8, products synthesized with Pol ,B alone; 9, conditions as in lane 2 except that RNase-treated 4X DNA-RNA hybrid was incubated with Pol a alone; 10, as in lane 2 but with Pol 13 alone. Markers were 0.2 ,ug each of OX DNA-RNA hybrid, OX RF II isolated from the OX RF H-A protein complex (23), and OX RF III generated by partial pancreatic DNase digestion of OX superhelical, double-stranded, closed circular DNA (RF I) (23); markers were visualized by ethidium bromide staining.

also used to quantitate the Pol activity (Table 1). Pol a in combination with Pol /3 catalyzed more than additive DNA synthesis with 46X DNA-RNA hybrid; Pol a or Pol ,3 alone utilized this RNA-primed DNA poorly. In the presence of both Pols and OiX DNA-RNA hybrid, deoxynucleotides were incorporated at a constant rate for 180 min at 300C. The morethan-additive synthesis of DNA catalyzed by the combined action of Pol a and Pol ,B was not observed with MVM DNA or poly(dT).oligo(rA). MVM DNA-primed synthesis was catalyzed by Pol / but poorly by Pol a. These results suggest that Pol a catalyzes RNA-primed DNA synthesis but does not support extensive elongation of DNA chains; in contrast, Pol ,B can extend DNA chains provided that the primer is DNA. The requirement for two enzymes in RNA-primed DNA synthesis was also supported by experiments with N-ethylmaleimide (MalNEt); treatment of Pol a or Pol /3 with MalNEt (10 mM) prior to DNA synthesis abolished the more-thanadditive incorporation observed with RNA-primed OX DNA. In these experiments, sensitivity of Pol /3 to MalNEt varied depending upon the template-primer. MalNEt-treated Pol ,B retained full activity with poly(rA).oligo(dT) and 25% of its activity with MVM DNA but was inactive with the RNAprimed template (in conjunction with Pol a). Pol y failed to utilize either DNA- or RNA-primed DNAs but was active with

poly(rA).oligo(dT).

Structure and Size of DNA Products Synthesized with #X DNA-RNA Hybrids as Template. DNA chain elongation catalyzed by Pol a in combination with Pol /3 resulted in the formation of RF II, double-stranded full-length linear OX174 DNA (RF III), and products that migrated between RF III and OX DNA-RNA hybrid (Fig. 1). Removal of the RNA primer with RNase A (2 ,ug/ml) plus RNase T1 (10 units/ml) markedly

Biochemistry: l

2

Ikeda et al. >

.1

.,

4~~~~~~ 6I\1 1rI 2

FIG. 2. Size of DNA synthesized by Pol a and Pol with OX DNA-RNA hybrid. The RF II, RF III, OX DNA-RNA template, and products that migrated between RF III and OX DNA-RNA template on a neutral. agarose gel were electrophoretically eluted from lane 1 of the gel shown in Fig. 1. The DNAs were concentrated by ethanol precipitation, dissolved in 0.3 M NaOH/1 mM EDTA, and incubated at 250C for 10 min; each DNA sample (t1500 cpm) was subjected to alkaline agarose gel electrophoresis. Radioautography was carried out as described in the legend to Fig. 1. DNA isolated from RF II, RF Ill, the area between RF III and XX DNA-RNA hybrid, and the OX DNA-RNA template regions were in lanes 1-4, respectively. The products formed by the action of Pol ft with XX DNA-RNA hybrid treated with RNases A and T1 (as described in Fig. 1, lane 2) and with MVM DNA (containing 0.26 and 6.1 pmol of dTMP, respectively) were subjected to alkaline denaturation and gel electrophoresis. Approximately 5000 cpm of each was used. Lane 5, product formed with RNase-treated 4X DNA-RNA hybrid; lane 6, product-formed with MVM DNA. Linear XX DNA (5375 nucleotides long) and OX RF I Hpa I fragments (3730, 1264, and 392 base pairs) were treated with 0.3 M NaOH, fractionated on the same gel, and visualized with acridine orange (25).

decreased RF II synthesis; small amounts of RF III were formed, presumably from linear OX DNA molecules present in the OX DNA-RNA hybrid preparations. Once DNA elongation had commenced, the addition of RNases had no effect. DNA chain elongation required Mg2+ and was virtually unaffected by ATP; aphidicolin (10,MM), an inhibitor of Pol a, partially inhibited the reaction.* Pol a alone or Pol f3 alone synthesized small amounts of DNA; the amounts were even less if RNases were present. Full-size RF II molecules formed with Pol a plus Pol (3 appeared after a lag period of 25-30 min and then accumulated at a constant rate for approximately 60-180 min. The amounts of RF II accumulated at 60 and 180 min were 20% and 37% of the total DNA synthesized, respectively. The DNA synthesized by Pol a plus Pol # with OX DNA. RNA hybrid was also analyzed as follows. RF II, RF III, and material migrating between RF III and OX DNA-RNA hybrid were extracted from neutral gels (Fig. 1), treated with 0.3 M NaOH to remove RNA, and then subjected to alkaline agarose gel electrophoresis (Fig. 2). The newly synthesized DNA isolated from the RF II region of the native gel was 4700 nucleotides long. This size approximates the length of full-size 4X DNA minus the length of the RNA primer. More than 80% of incorporated asp was recovered as DNA 4700 nucleotides long. * The inhibitory effects of aphidicolin depend upon the concentration

of dNTPs present. The partial inhibition observed was more pronounced with lower levels of dNTPs.

Proc. Natl. Acad. Sci. USA 77 (1980)

5829

[32P]DNA isolated from regions of the gel corresponding to material migrating between RF III and OX DNA-RNA hybrid varied widely in length; the largest DNA detected was about twice the rX174 genomic length (Q9000 nucleotides long). DNA chains larger than the original OX DNA template were products formed by Pol # rather than Pol a; these DNA products most likely were chains extended from hairpin regions on linear OX DNA molecules. Experiments in which OX DNARNA hybrid preparations pretreated with RNase A and RNase TI were used (Fig. 2, lane 5) yielded long OX DNA products with Pol # but not with Pol ca (data not shown). DNA synthesis catalyzed by Pol # with MVM DNA yielded products nearly twice the length of MVM DNA (-u8000 nucleotides). Thus, Pol # can extensively elongate DNA primers hydrogen bonded to long single-stranded DNA templates. How Far Does Pol a Extend an RNA Primer? The OX DNA-RNA template was incubated with Pol a, dNTPs, and [a-32P]dTTP for 10 min and then chased as indicated. The product was treated with 0.3 M NaOH and analyzed on an alkaline agarose gel (Fig. 3). The DNA formed was 230 nucleotides long and was partially elongated to a maximum length of 390 nucleotides by addition of more Pol a or a more prolonged incubation. Sequential Roles of Pol a and Pol P. OX DNA-RNA hybrid was incubated with Pol a, dNTPs, and [a-32P]dTTP for 10 min and the labeled product was isolated. This 32P-labeled product was incubated with Pol a or Pol # or both in the presence of Unlabeled dNTPs; the products were then analyzed on a neutral agarose gel (Fig. 4). During the first incubation, Pol a elongated the RNA primer with deoxynucleotides. In the second reaction, Pol a hardly affected the length of the DNA chain covalently attached to RNA primer but Pol ,3 or the combination of Pol

2v

H)

--a-

FIG. 3. Size of DNA synthesized on XX DNA-RNA hybrid with Pol a. The complete system (0.4 ml) included 1.6 pg of OX DNA-RNA hybrid and 0.94 ,g of Pol a with 10 $M [a-32P]dTTP (20,000 cpm/ pmol) as the labeled dNTP. After 10 min at 300C, unlabeled dTTP was added to yield a final dTTP concentration of 2 mM. The mixture was divided into 0.05-ml aliquots (each containing 0.68 pmol of acid-insoluble dTMP) and incubated at 300C for 50-110 min with or without additional Pol a (118 ng). 32P-Labeled products were isolated, ethanol precipitated, and treated with 0.3 M NaOH. Approximately 10,000 cpm of each product was fractionated on an alkaline agarose gel (45 V for 3.5 hr) and radioautographed as described in Fig. 1. Lanes: 1, products formed after 10-min incubation; 2, products formed after 50-min incubation; 3, products formed after 110-min incubation; 4, products formed after 110-min incubation, with additional Pol a (117 ng) added at the beginning of the long incubation period. OX RF I Hpa I fragments (1265 and 392 base pairs) and XX RF I HindI fragments (297, 210, and 162 base pairs) were treated with 0.3 M NaOH and used as markers.

5830

Proc. Natl. Acad. Sci. USA 77 (1980)

Biochemistry: Ikeda et al.

Table 2. Effects of Pol on in vitro replication of Ad DNA-pro dTMP incorp., pmol Additions

600 600-

BPB

400 20010

40 50 60 70 80 90 Fraction FIG. 4. Elongation of newly synthesized DNA covalently attached to an RNA primer. An incubation mixture (0.5 ml) containing 1 ,g of XX DNA-RNA hybrid was incubated with 1 ,g of Pol a for 10 min at 30'C and the reaction was halted by the addition of 10 mM EDTA. The products, 2.4 pmol labeled with [a-32P] dTTP (15,000 cpm/pmol), were treated with phenol, concentrated by ethanol precipitation, and dissolved in 10 mM Tris-HCl, pH 7.5/1 mM EDTA/10 mM NaCl. The isolated products (150 ng of DNA, 6000 cpm) were incubated in the complete system (0.05 ml) in the presence of unlabeled dNTPs with no enzyme (0), 130 ng of Pol a (0), 80 ng of Pol /3 (A), or 130 ng of Pol a plus 80 ng of Pol /3 (4) at 30°C for 60 min. Approximately 3000 cpm of each product was isolated, concentrated, and fractionated on neutral agarose gel. Gels were sliced into 1-mm sections, dissolved with 10 ml of Aquasol in glass vials, and assayed for radioactivity. 20

30

a and Pol / fully extended the 32P-labeled product to the RF II structure. Pol y, alone or in combination with Pol a, did not elongate products formed in the first incubation step. Pol y had no effect on the action of Pol / in generating RF II products (data not shown). Effects of HeLa Cell DNA Polymerases on the in Vitro Replication of Ad DNA. The in vitro replication of Ad DNA provides a means for the identification and isolation of proteins that play an important role in this viral system. This in vitro system (21, 22) has been resolved into fractions derived from

the cytosol of Ad virus-infected HeLa cells and fractions isolated from nuclei; the latter fractions can be prepared from uninfected HeLa or Chinese hamster ovary cells. Ad DNA synthesis is absolutely dependent on the four dNTPs, Mg2+, ATP, and Ad DNA-pro. Ad DNA-dependent DNA synthesis with cytosol from infected cells plus nuclear extracts yields full-sized Ad DNA (unpublished data). The activity contributed by nuclear extracts from uninfected cells could be replaced with purified Pol ,B but not with Pol a or Pol y (Table 2). This reaction was also dependent upon addition of ATP and required the Ad DNA-pro complex and yielded full-length Ad DNA products (data not shown). Although MalNEt-treated Pol ,B showed no loss of activity with poly(rA) oligo(dT) as primer template, MaINEt treatment of Pol / or nuclear extracts prior to synthesis on Ad DNA-pro completely inhibited their complementing activity in the viral system. The addition of Pol a and Pol y to cytosol fractions did not enhance DNA synthesis above that detected with Ad-infected cytosol alone. DISCUSSION These results show that the three eukaryotic Pols utilized RNA-primed OX DNA poorly; only Pol / utilized DNA-primed MVM effectively. These experiments were carried out with low concentrations of primer termini. The combination of Pol a and Pol / was essential for synthesis of long DNA products with RNA-primed OX DNA. In agreement with results of Keller (27) and Spadari and Weissbach (28), Pol a catalyzed the limited

7.48 Cytosol + nuclear extract (complete) 1.29 Cytosol alone 0.59 Nuclear extract alone 1.38 Cytosol + nuclear extract* 0.44 Cytosol* + nuclear extract 1.57 Cytosol + Pol a (0.059 unit) Cytosol + Pol /: 3.88 0.011 unit 5.81 0.028 unit 5.87 0.046 unit 1.10 Cytosol + Pol /3* (46 units) 1.21 Cytosol + Pol -y (41 units) Reaction mixtures (0.05 ml), containing 50 mM Hepes buffer (pH 7.5), 0.5 mM dithiothreitol, 5 mM MgCl2, 3.8 mM ATP, dATP, dGTP, and dCTP at 50MgM each, 8MM [3H]dTTP (3800 cpm/pmol), and 0.15 gg of Ad DNA-pro, were incubated at 37°C for 60 min. Reactions were carried out with Ad-infected HeLa cell cytosol (70 ,g), uninfected Chinese hamster ovary cell nuclear extract (26 ,g), and Pol as indicated. The cytosol preparation was a 25-60% ammonium sulfate saturated fraction prepared from the cytoplasm of Ad-infected HeLa cells (21). Nuclear extracts were prepared from nuclei of uninfected Chinese hamster ovary cells as described (22). Each fraction was assayed independently for its Pol activity. In the presence of nicked salmon sperm DNA, incorporation with cytosol (68.6 pmol) and nuclear extracts (16.8 pmol) was decreased to 4.9 and 3.5 pmol, respectively, with 20 mM MalNEt. With poly(rA).oligo(dT), incorporation with the cytosol fraction (10.4 pmol) and nuclear extract (2.4 pmol) was 1.6 and 2.6 pmol, respectively, after MalNEt treatment. In these experiments, varying the concentrations of Pol a or Pol y had no further effect; the combination of purified polymerases did not alter the results obtained with Pol /3 alone. Omission of DNA in all experiments decreased dTMP incorporation to 0.1 pmol which has not been subtracted from the above results. Although the 1.3 pmol of dTMP incorporated by infected cytosol fractions alone was completely dependent on addition of Ad DNA-pro, only 10% of the product was full-length viral DNA. * Treated with MalNEt.

synthesis of chains approximately 230 nucleotides long on RNA-primed DNA templates. The present results show that these short DNA chains could be elongated by Pol / to RF II (z4500 nucleotides long). Under the conditions used, Pol y was inactive with. all the primer-template systems other than the synthetic poly(rA)-oligo(dT). At present, the enzymes involved in the elongation of primed templates in eukaryotic systems are unknown. Until specific mutants conditionally blocked in replication are discovered, this problem will be difficult to resolve. The availability of the in vitro replication system for Ad DNA may be helpful in characterizing cellular proteins involved in DNA replication. To date, no new Pol has been found after this viral infection (29). We have found that Pol /3 can participate in the in vitro synthesis of Ad DNA, presumably complementing the abundant amount of Pol a found in the cytoplasm. This is in keeping with the ability of Pol / to elongate short DNA primers extensively. In prokaryotes, the elongation of primed templates is dependent upon at least three proteins in addition to Pol III (24, 30). In the absence of these proteins, Pol II and Pol III of E. coli only catalyze a short extension of primed templates (31).- In this respect, Pol a is similar to these bacterial polymerases. In light of the complicated manner by which prokaryotes catalyze elongation of primed DNA templates, it is not surprising to note the number of different protein factors already reported that alter the properties of Pol a and /3(1, 32-34). However, unlike the prokaryotic system, our results suggest the need for more

Proc. Natl. Acad. Sci. USA 77 (1980)

Biochemistry: Ikeda et al. than one eukaryotic Pol for the elongation at individual replication forks. Further studies are needed to clarify the effects of MalNEt on Pol ,B. The inhibition of this enzyme activity by MaINEt was detected with templates containing low concentrations of primer ends. Whether this reflects the presence of additional factors or different functional sites on the enzyme remains to be elucidated. Attempts to define the nature of the Pols involved in replication by the use of inhibitors such as ddTTP, aphidicolin, and MaINEt represent at best an approximation. Studies with aphidicolin showed that this tetracyclic diterpenoid does not completely inhibit Pol in vitro, although it is a potent inhibitor of DNA replication in vivo (7, 9, 10). This suggests that additional factors may be involved. Because the incorporation of ddTTP causes chain termination, the effect of this inhibitor will depend upon the number of ends available, the ratio of dTTP to ddTTP, and the presence of exonucleases capable of hydrolyzing the incorporated ddTMP at the 3'-hydroxyl end. The effects of MalNEt, as shown here, depend upon the assay system used. Although our results suggest an important role for Pol in the replication of Ad DNA, it is not clear that this is indeed the case in viw. We and others (6-9) have reported the importance of Pol and Pol y in Ad DNA replication. Because the cytosol fraction from Ad-infected cells contained Pol a and Pol we could not evaluate the role of these enzymes by their addition to the Ad DNA replication system. Further studies involving the resolution of the proteins essential for Ad DNA replication should help to clarify some of these problems. a

a

This work was supported by Grant 5R01 GM13344-15 from the National Institutes of Health, Grants 5RO1 CA 21622-04 and CA-11512 from the National Cancer Institute, and Grant NP89L from the American Cancer Society. 1. Weissbach, A. (1977) Annu. Rev. Biochem. 46,25-47. 2. Edenberg, H., Anderson, S. & DePamphilis, M. L. (1978) J. Biol. Chem. 253, 3273-3280. 3. Otto, B. & Fanning, E. (1978) Nucleic Acids Res. 5, 17151728. 4. Waqar, M., Evans, M. & Huberman, J. (1978) Nucleic Acids Res. 5, 1933-1946. 5. Hubscher, U., Kuenzle, C. C. & Spadari, S. (1979) Proc. Natl.

Acad. Sci. USA 76,2316-2320. 6. van der Vliet, P. C. & Kwant, M. M. (1978) Nature (London) 276, 532-534. 7. Krokan, H., Schaffer, P. & DePamphilis, M. L. (1979) Biochemistry 18, 4431-4443.

5831

8. Abboud, M. M. & Horwitz, M. S. (1979) Nucleic Acids Res. 6,

1025-1039. 9. Longiaru, M., Ikeda, J.-E., Jarkovsky, Z., Horwitz, S. B. & Horwitz, M. S. (1979) Nucleic Acids Res. 6,3369-3386. 10. Oguro, M., Hori, C., Nagano, H., Mano, Y. & Ikegami, S. (1979) Eur. J. Biochem. 97,603-607. 11. Wist, E. & Prydz, H. (1979) Nucleic Acids. Res. 6, 1583-1590. 12. Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H. & Mano, Y. (1978) Nature (London) 275,458-460. 13. Ohashi, M., Taguchi, T. & Ikegami, S. (1978) Biochem. Biophys. Res. Commun. 82,1084-1090. 14. Bertazzoni, A. & Spadari, S. (1976) Proc. Natl. Acad. Sci. USA 73,785-789. 15. Eichler, D. C., Wang, T. S.-H., Clayton, D. A. & Kom, D. (1977)

J. Biol. Chem. 252,7888-7893.

16. Bolden, A., Pedrali-Noy, G. & Weissbach, A. (1977) J. Biol. Chem.

252,3351-3356.

17. Bertazzoni, U., Scovassi, A. I. & Brun, G. (1977) Eur. J. Biochem. 81,237-248. 18. Hubscher, U., Kuenzle, C. C. & Spadari, S. (1977) Eur. J. Biochem. 81, 249-258. 19. Karkas, J. D. & Chargaff, E. (1966) Proc. Natl. Acad. Sci. USA 56, 1241-1246. 20. Kornberg, A. (1980) DNA Replication (Freeman, San Fran-

cisco).

21. Challberg, M. D. & Kelly, T. J., Jr. (1979) Proc. Nati. Acad. Sci. USA 76,655-659. 22. Kaplan, L. M., Ariga, H., Hurwitz, J. & Horwitz, M. S. (1979) Proc. Natl. Acad. Sci. USA 76,5534-5538. 23. Ikeda, J.-E., Yudelevich, A. & Hurwitz, J. (1976) Proc. Nati. Acad. Sci. USA 73,2669-2673. 24. Hurwitz, J. & Wickner, S. (1974) Proc. Nati. Acad. Sci. USA 71, 6-10. 25. McMaster, G. K. & Carmichael, G. G. (1977) Proc. Nati. Acad. Sci. USA 74,4835-4838. 26. Vicuna, R., Hurwitz, J., Wallace, S. & Girard, M. (1977) J. Biol.

Chem. 252,2524-2533 27. Keller, W. (1972) Proc. Natl. Acad. Sci. USA 69, 1560-1564. 28. Spadari, S. & Weissbach, A. (1975) Proc. Natl. Acad. Sci. USA

72,503-507. 29. Ito, K., Arens, M. & Green, M. (1975) J. Virol. 15, 1507-1510. 30. Scheckman, R., Weiner, J. J. & Kornberg, A. (1974) Science 186, 987-993. 31. Wickner, R. B., Ginsberg, B. & Hurwitz, J. (1972) J. Biol; Chem.

247,498-504. 32. Mosbaugh, D. W., Stalker, D. M., Probst, G. S. & Meyer, R. R. (1977) Biochemistry 16, 1512-1518. 33. Novak, B. & Baril, E. F. (1978) Nucleic Acids Res. 5, 221-239. 34. Blue, W. T. & Weissbach, A. (1978) Biochem. Biophys. Res. Cormmun. 84, 603-610.