Proc. Nati. Acad. Sd. USA Vol. 76, No. 11, pp. 5534-5538, November 1979 Biochemistry
Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro (eukaryotic DNA replication/soluble nuclear extracts/DNA binding -protein)
LEE M. KAPLAN, HIROYOSHI ARIGA, JERARD HURWITZ, AND MARSHALL S. HORWITZ Departments of Cell Biology, Microbiology and Immunology, Developmental Biology and Cancer, and Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461
Contributed by Jerard Hurwitz, August 7,1979
ABSTRACT Soluble extracts of adenovirus-infected HeLa cell nuclei support DNA replication on exogenous adenovirus DNA templates. Conditions of synthesis using both wild-type and temperature-sensitive extracts have been defined. Nuclear extracts prepared from cells permissively infected with the adenovirus mutant H5ts125 expressed the temperature-sensitive phenotype and could be inactivated at 370C in vitro. These extracts were completely complemented by the addition of wildtype adenovirus DNA binding protein but not by H5ts125 DNA binding protein. Enhancement by binding protein in the mutant extracts represents replication, as demonstrated by the production of full-sized products and orderly chain elongation originating, as in vivo, at both ends of the linear DNA. Replicative synthesis required the 5'-terminal protein bound covalently to template DNA and could be inhibited by denaturation of this 55,000-dalton protein. Various inhibitors of eukaryotic DNA polymerases, such as aphidicolin and 2',3'-dideoxythymidine triphosphate, inhibited replication of exogenous adenovirus templates in this system as they do in previously reported systems that only elongate endogenous replicating intermediates. Challberg and Kelly (1) have recently shown that nuclear extracts of adenovirus type 5 (Ad5)infected HeLa cells catalyzed the semiconservative replication of exogeneously added Ad DNA. DNA synthesis on a template of AdS DNA covalently bound to the 55,00-dalton 5'-terminal protein (Ad5 DNA-Pro) closely resembled synthesis in vvo; however synthesis on deproteinized Ad5 DNA represented a repair-like reaction (1, 2). This system, which presumably initiates and elongates DNA on exogeneously added template, should permit the fractionation and characterization of components required for viral DNA replication. Studies in prokaryotes have depended on the use of conditional-lethal mutants in genes involved in DNA synthesis and the specific inactivation of these genes in cell extracts (3-6). Although analogous mutants in eukaryotic cells have been difficult to obtain, there are three complementation groups of conditional-lethal DNA-negative Ad mutants (7). The defect of one such AdS mutant, H1tsl25, was identified as the gene coding for a 72,000-dalton polypeptide that binds to singlestranded DNA (8, 9). The mutant is temperature-sensitive for viral DNA replication (10), and the DNA binding protein (DBP), isolated from H5ts125-infected cells at permissive temperature, exhibits a temperature-dependent binding to single-stranded DNA cellulose columns that is different from that of the wild-type protein (9). Studies with whole cells infected with H5ts125 and with nuclei prepared from those cells have suggested that the defect in DNA synthesis is due to a failure of initiation (11, 12). Studies with ammonium sulfate nuclear extracts of such cells, which catalyze only the elongation of replicating intermediates previously initiated in vivo (13),
have shown that funtional DBP is also required for the elongation reaction (14). This communication describes further studies of the replication reaction on added exogenous viral DNA catalyzed by nuclear extracts from infected cells. E. tracts prepared from H5tsI25-infected cells were thermolabile and could be inactivated by preincubation at 370C. Under these conditions, full activity was reconstituted by the addition of wild-type Ad2 DBP which generated a reaction having all the characteristics of true replication. We have confirmed that replicative DNA synthesis required viral DNA bound to the 5'-terminal protein (2). Denaturation of this protein inactivated the template activity of the DNA-protein complex. Thus, dependence of DNA replication in vitro on specific viral-coded and virus-associated proteins suggests that the system will allow further study of these and other requirements for DNA replication in eukaryotic systems.
The publication costs of this article were defrayed in part by page
Abbreviations: Ad5, adenovirus type 5; Ad DNA-Pro, adenovirus DNA covalently bound to a 55,000-dalton protein at the 5'-end of each strand; H5ts125, an AdS temperature-sensitive mutant in viral DNA
MATERIALS AND METHODS Cells and Viruses. The sources of HeLa S3 cells, Ad2, and H5ts125 as well as the growth of cells, the growth and purification of stock virions, and infections for nuclear extracts were as described (14, 15). Preparation and Extraction of Infected Nuclei. Cells infected with Ad2 were grown at 370C; those infected with H5ts125 were grown at 32.50C. Two to 4 hr after infection, hydroxyurea was added (final concentration, 3-10 mM), and infections were continued for a total of 18-22 hr for Ad2 and 40 hr for H5ts125. At these times, cells were washed and nuclei were prepared according to the procedure of Challberg and Kelly (1). Nuclei were frozen in liquid nitrogen and stored at -80'C. Frozen nuclei were thawed at 0WC, various amounts of 5 M NaCl were added, and the nuclei were incubated at 0C as described for each experiment. The extracted nuclei and nuclear debris were then removed by centrifugation for 20 min at 15,000 X g in a Beckman type 40 rotor. Preparation of Labeled Viral DNA, DNA-Pro, and DBP. ["4C]Thymidine-labeled Ad2 virion and uniformly "4C-labeled Ad2 deproteinized DNA were prepared as described (16). Ad2 ["4C]DNA-Pro was purified from virions by the method of Sharp et al. (17) and dialyzed against five changes (200 vol each) of 10 mM Tris-HCI, pH 8.1/1 mM EDTA at 40C. [ 5S]Methionine-labeled DBP was purified from HeLa cells infected with either Ad2 or H5ts125 and the purity of these preparations was determined as described (14). Ad2 DBP used in these experiments was approximately 90% pure, and H5ts125 DBP was approximately 70% pure.
charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
synthesis; DBP, viral-coded DNA binding protein. 5534
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formed, it was completely solubilized by DNase I at 100
Synthesis of DNA In Vitro. Infected nuclear extracts were incubated in reaction mixtures (0.1 ml) containing-s0 mM Hepes (pH 7.2), 1 mM dithiothreitol, 7.5 mM MgCI2, 2 mM ATP, 50 MM each dATP, dCTP, and dGTP, 5,uM [3H]dTTP (1 X 104 cpm/pmol), and 0.15-0.2 jig of DNA or DNA-Pro template (450-600 pmol of nucleotides). Purified Ad2 or H5ts125 DBP was added to reactions as indicated. For those H5ts125 reactions in which preincubation was carried out, nuclear extracts were incubated in a mixture (80 Al) containing 62.5 mM Hepes (pH 7.2), 0.63 mM dithiothreitol, 6.25 mM MgCI2, 2.5 mM ATP, and DBP as indicated. After preincubation, DNA synthesis was started by the addition of 20 ,u of a solution containing dATP, dCTP, and dGTP each at 250,M, 15 ,M [3H]dTTP, and 450 pmol of Ad2 DNA-Pro. The temperature of preincubations and DNA synthesis reactions was 30 or 370C, as indicated. After incubation, acid-insoluble radioactivity was quantitated as described (13). For nuclease analysis of the product, reactions were stopped by the addition of 1 M NaCI and diluted 1:10 with 20 mM Tris-HCI, pH 7.5/1 mM dithiothreitol/7.5 mM MgGI2; enzymes were added, and the samples were incubated for 30 min at 37°C. Materials. Protein concentrations were determined by the method of Bradford (18) using stock solutions of the proteinbinding dye reagent purchased from Bio-Rad. Hydroxyurea was obtained from either Squibb or Aldrich. All other reagents were as described (13, 14). RESULTS DNA Synthesis Catalyzed by Nuclear Extracts. Nuclear extracts prepared by treating Ad2-infected nuclei with 200 mM NaCl for 60 min were analyzed for their ability to catalyze Ad2 DNA-Pro-directed DNA synthesis (Table 1). The requirements for synthesis in vitro were as described (1). Maximal incorporation of dTMP, using 600 pmol (0.2 Mg) of DNA-Pro template and 25 Ml of extracts (120 Mig of protein), was 7.2 pmol in 1 hr. DNA synthesis required all four dNTPs and Mg+2 in addition to Ad2 DNA-Pro and was stimulated 4-fold by the addition of ATP. Inclusion of all four rNTPs had little effect on the reaction. The reaction was inhibited by the addition of DNase I, a mixture of RNase A and RNase T1, or proteinase K. There was no inhibition by RNase H, and only partial inhibition by the single-strand-specific Neurospora nuclease. Once the product
ug/ml) plus RNase Ti (20 units/ml). Effect of Various Polymerase Inhibitors on Ad2 DNA Synthesis In Vitro. We examined the effect of several inhibitors on DNA replication in the reaction catalyzed by Ad2 nuclear extracts (Table 2). Aphidicolin, which has been shown to inhibit Ad DNA replication in vio and specifically to inhibit DNA polymerase acby 95% at 100,uM (20-22), inhibited replication catalyzed by nuclear extracts. 2',,3'-Dideoxythymidine triphosphate also inhibited synthesis in vitro under conditions at which DNA polymerase y is inhibited but DNA polymerase is completely unaffected (23,24). Actinomycin D inhibited the reaction >90% at 10,Mg/ml. a-Amanitin, a specific inhibitor of RNA polymerase II, had no effect on this reaction, even at concentrations higher than those which will completely inhibit mRNA synthesis (25). Differentiation Between Replicative and Nonreplicative DNA Synthesis In Vitro. We examined the activity of Ad2 nuclear extracts on both Ad2 DNA-Pro and Ad2 DNA templates as a function of the NaCI concentration used to make the extracts (Fig. 1A). Low-salt (0-50 mM) extracts catalyzed similar dTMP incorporation with Ad2 DNA-Pro or DNA but the size of both products on alkaline sucrose gradients was small, suggesting nonreplicative synthesis. High-salt extracts showed a greater discrimination between the two templates and the discrimination with 200 mM extracts was nearly complete. Activity of the Ad2 nuclear extracts was completely dependent on exogenous DNA templates. The product synthesized by 25 ul of the 200 mM Ad2 wild-type nuclear extracts on Ad2 DNA-Pro was full-sized on alkaline and neutral sucrose gradients, and replication originated at both ends of the linear DNA template (data not shown). Therefore, synthesis of fulllength DNA and, particularly, discrimination between the Ad DNA-Pro and Ad DNA templates depended on factors that were extracted in sufficient quantity only by treating nuclei with relatively high (100-200 mM) concentrations of NaCl. With 200 mM nuclear extracts, replicative synthesis required a concentration of extracts greater than that needed for maximal dTMP incorporation (Fig. 1B). Reaction mixtures containing 5 ,l of extracts (25 MAg of protein) incorporated dTMP equally well with Ad2 DNA-Pro and Ad2 DNA templates.
Table 1. Requirements for Ad2 DNA synthesis catalyzed by Ad2 nuclear extracts dTMP incorporated, Conditions pmol/15 min 2.36 Complete - Ad2 DNA-Pro 0.11 - MgCl2 0.08
Table 2. Effect of inhibitors on Ad2 DNA synthesis catalyzed by nuclear extracts dTMP incorporated, % activity Additions pmol/15 min 1.74 None 100 Aphidicolin 10MM 1.32 76
-ATP
0.57
0.29 dATP, dCTP, dGTP + CTP, GTP, UTP (50,uM) 2.60 + DNase I: 50,ug/ml 0.63 100 ug/ml 0.10 + Neurospora nuclease (0.3 unit/ml) 1.85 + RNase A (50,ug/ml), RNase T1 0.17 (10 units/ml) + RNase H (10 units/ml) 2.61 The complete reaction incubated at 370C was as described in Materials and Methods except that the unlabeled dNTPs used were treated with NaIO4 and repurified by DEAE-Sephadex chromatography to remove trace contaminants of ribonucleotides (19). Reaction mixtures contained 600 pmol of Ad2 DNA-Pro template and 25 ,l of nuclear extracts (120 ,g of protein). -
was
,ag/ml but was then unaffected by treatment with RNase A (100
a
100MOM 2',3'-Dideoxythymidine triphosphate 5,MM 15MAM Actinomycin D 1 g/iml 10Mug/im
0.21
12
0.26 0.15
15 9
0.84 0.13
48 7
a-Amanitin
20Mug/ml 50Mug/ml
1.75 101 1.36 78 DNA synthesis reactions were as described in Table 1. Actinomycin D was dissolved in ethanol; aphidicolin was dissolved in dimethyl sulfoxide. These organic solvents were present in the reaction mixtures at concentrations of 1%, which did not affect dTMP incorpo-
ration.
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Proc. Natl. Acad. Sci. USA 76 (1979)
-Q~~~~3-4
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03
33
2-
0
1 00 200 NaCI, mM
5
15
Extract, AI
25
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15
5
Extract, Ail
FIG. 1. Differentiation between replicative and nonreplicative dNMP incorporation. (A) Effect of NaCl concentration on extraction of infected nuclei. Ad2-Infected nuclei were prepared and thawed at 00C, aliquots were extracted for 60 min with various concentrations of NaCl (0-200 mM), and activity was measured. Incorporation of [3H]dTMP into acid-precipitable material was measured by using 600 pmol (0.2 pg) of either Ad2 DNA-Pro (-*0) or Ad2 DNA (o -- o) as template in the presence of 25 sl of nuclear extracts (100-120 ,.g of protein). Reactions were at 371C for 30 min. (B) Effect of concentration of nuclear extract. The activity of various concentrations of Ad2-infected nuclear extracts prepared with 200 mM NaCl was measured on Ad2 DNA-Pro (0-0) or Ad2 DNA (O -0). Conditions for extraction and reaction were as in A. (C) Temperature sensitivity of H5ts125 nuclear extracts. H5ts125-infected nuclei were extracted with 100 mM NaCl for 5 min. Activity in various concentrations of extracts was measured for 60 min at either 300C (A-A) or 370C (AA) after a 60-min preincubation at the same temperature; 25 sAl of nuclear extract contained 120 ug of protein. -
---
- -
from cells infected with either Ad2 at 370C or HSts125 at 32.50C (permissive temperature). Various amounts of each DBP were added to the H5ts125 nuclear extracts, the mixtures were preincubated for 60 min at 370C, and DNA synthesis was then assayed (Fig. 2). Purified Ad2 DBP complemented the temperature-sensitive defect, yielding 133% of wild-type activity with 3.2 ,ug of DBP. In contrast, similar concentrations of temperature-sensitive DBP purified from H5ts125 infected cells were unable to complement the defect at 370C. Mixtures of Ad2 DBP and H5ts125 DBP complemented at the level of wild-type DBP used, indicating that the inability of the mutant DBP to complement was not due to an inhibitor in the preparation. Thus, functional DBP is required for DNA synthesis catalyzed by these nuclear extracts. DBP purified from Escherichia colh was previously shown (26) to substitute partially for Ad2 DBP in the DNA elongation reaction on endogenous replicating intermediates. In contrast, E. cohi DBP was unable to substitute for Ad DBP in these extracts which required exogenous DNA. Characterization of the DNA Synthesis Reaction Products. The size and amount of DNA synthesized by H5ts125 nuclear extracts at 300C (permissive temperature), at 370C (nonpermissive temperature), and at 370C in the presence of Ad2 DBP were measured. After a 60-min preincubation and a 60-min reaction at 300C, nearly all of the DNA product sedimented as full-sized Ad DNA in both alkaline (Fig. 3A) and neutral (Fig. 3B) sucrose gradients. When the preincubation and the DNA synthesis reaction were at 370C, dTMP incorporation decreased significantly, although the residual product also sedimented as full-sized viral DNA. Most importantly, when the 370C preincubation and reaction were carried out in the presence of Ad2 DBP, full-length products were formed with
However, the products of reactions with S or 10 ,l of nuclear
extracts on alkaline sucrose gradients were small. Increasing the amount of nuclear extracts to 25 ,l (120 Mg of protein) re-
sulted in the production of full-sized Ad DNA and concurrently generated 20- to 50-fold discrimination between the two templates. Therefore, the DNA replication catalyzed by these extracts was distinct from a repair-type dTMP incorporation and required the presence of Ad DNA covalently bound to the 5'-terminal protein. Ad2 DNA-Pro was inactivated more than 85% by extraction three times with chloroform/isoamyl alcohol, 24:1 (vol/vol). Heating the Ad2 DNA-Pro to 70° in 10 mM Tris-HCl, pH 7.5/1 mM EDTA/10 mM NaCl for 15 or 60 min decreased the activity by approximately 55%. Mixing experiments showed that there were no inhibitory factors in any of the inactive DNA preparations described, and none of these results could be reproduced simply by varying the amount of NaCl in the reaction. Discrimination between replicative and nonreplicative DNA synthesis was also correlated with the thermolability of H5ts125 nuclear extracts. At low concentrations of extract (5 Ml), there was little inactivation of incorporation on Ad2 DNA-Pro templates at 370C (Fig. IC). Under these conditions, the products of the reaction at both 30 and 370C were small on alkaline sucrose gradients. However, with 25 Mul (120 Mug) of nuclear extracts, synthesis was inhibited more than 90% at 370C compared to controls incubated at 300C. At this concentration of extracts, synthesis at 30'C was replicative, as described below. These data suggest that, in addition to decreasing nonspecific incorporation on deproteinized DNA, higher amounts of extracts provided additional activities essential for replicative synthesis. Complementation of the H5ts125 Defect in DNA Replication by Ad2 DBP. Purified wild-type Ad DBP restored the activity of H5ts125 nuclear extracts at 370C. DBP was purified
8
a
'56 E
0
Q., C
2Ho
0
1
2
3
DBP, mg of FIG. 2. Complementation H5ts125-infected nuclear extracts by various DBPs. Various amounts of DBP purified from HeLa cells infected with Ad2 (0-0) or H5ts125 (O- - -0) or from Escherichia coli (A-A) were added to 25 IAI of nuclear extracts prepared from H5ts125-infected cells, and the mixtures were preincubated for 60 min at 370C. Reactions were begun by the addition of [3HldTTP, dATP, dCTP, dGTP, and Ad2 DNA-Pro. H5ts125 nuclear extracts were similarly treated at 300C without the addition of DBP (-). The incorporation of [3HJdTMP into acid-precipiteble material was measured after a 60-min reaction; 25 .l of nuclear extracts contained 120 ,ug of protein.
Biochemistry: Kaplan et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
5537
0
E 10
~0.6
U
3-
5
0.4 0.2
5
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15 Fraction
5
10
~
-
15
FIG. 3. Sedimentation analysis of DNA synthesized by H5ts125 nuclear extracts in the absence or presence of added Ad2 DBP. The direction of sedimentation in these gradients is from right to left. Aliquots (25 tl) of H5ts125 nuclear extracts containing 120 jug of protein were preincubated for 60 min at 30'C (0-0), at 370C (@--- *), or at 370C in the presence of 1.5 ug of Ad2 DBP (A-A). Reactions were begun by the addition of [3H]dTTP, dATP, dCTP, dGTP, and Ad2 DNA-Pro; incubation was for 120 min at the same temperature as the preincubation. (A) The reactions were terminated by the addition of 0.4 ml of 0.1 M EDTA/0.15 M NaCi at 0C. After removal of 0.1 ml for quantitation of dTMP incorporation, the remaining 0.4 ml was layered onto 5-201% alkaline sucrose gradients and centrifuged at 24,000 rpm in a Beckman SW 27.1 rotor for 16 hr at 4VC. The gradient containing the 301C reaction also contained 5 X 103 cpm of Ad2 [14CJDNA, added as virion before centrifugation. The arrow represents the position of the 34S marker Ad [14C]DNA single strands. Recoveries of 3H were 98% for the 300C reaction, 80% for the 370C reaction, and 93% for the 370C reaction with Ad2 DBP. The pellets of these gradients contained 51, 129, and 79 cpm, respectively. (B) The reactions were stopped with 0.4 ml of 0.5% sodium dodecyl sulfate/10 mM EDTA/50 mM NaCl/10 mM Tris.HCl, pH 7.4. Aliquots (0.4 ml) were layered onto 5-20% neutral sucrose gradients containing 0.5% sodium dodecyl sulfate and centrifuged at 14,000 rpm in a Beckman SW 27.1 rotor for 16 hr at 25°C. The gradient containing the 30°C reaction also contained 5 X 103 cpm of Ad2 [14CJDNA, added before centrifugation; the arrow represents the position of this 31S marker. Recoveries of 3H were 85% for the 30°C reaction, 75% for the 37°C reaction, and 92% for the 37°C reaction with Ad2 DBP. The pellets of these gradients contained 70, 87, and 134 cpm, respectively.
identical sedimentation characteristics as those formed at 300C. Studies with intact cells have shown that Ad DNA replication occurs from both ends of the linear duplex molecules (27-30). Replication in vttro also begins at or near either end of the template as demonstrated by the observation that the terminal restriction fragments of the product are preferentially labeled at early times in the course of this reaction. Only after longer reactions is the distribution of label across the genome more uniform (2). We have confirmed these observations with 200 mM NaCl nuclear extracts of Ad2-infected cells at 370C. To characterize the DNA products synthesized by H5ts125 extracts complemented by Ad2 DBP at 37'C, we used a similar analysis. After preincubation of H5ts125 nuclear extracts in the presence of Ad2 DBP for 60 min at 37°C, DNA synthesis was initiated by the addition of Ad2 DNA-Pro and the four dNTPs. Aliquots were removed at intervals for the subsequent 40 min, and the DNA was purified and digested with Hpa I. The DNA reaction product formed during the first 5 min of incubation was labeled primarily in the terminal fragments (Fig. 4). With increasing time, more label was detected in internal fragments and, after 40 min, the distribution of label across the genome was essentially uniform. Ad DNA synthesized by the H5ts125 nuclear
O
E
C
F
A
B
D G
FIG. 4. Kinetics of labeling of various regions of DNA synthesized by H5ts125 nuclear extracts in the presence of added Ad2 DBP. H5ts125 nuclear extracts (0.25 ml) were preincubated for 60 min at 37°C in the presence of 8 ,ug of Ad2 DBP; the reactions were begun by addition of [3H]dTTP, dATP, dCTP, dGTP, and Ad2 DNA-Pro. Reactions were stopped after 5, 10, or 40 min by the addition of 20 mM EDTA at 0°C. Each aliquot was digested with proteinase K (500 wu/ml) for 30 min at 37°C. The samples were centrifuged in 5-20%o sucrose gradients (11 ml) in 0.5 M NaCl/20 mM Tris.HC1, pH 7.4/10 mM EDTA at 38,000 rpm (Beckman SW 41 rotor) for 2.5 hr at 15°C. All fractions containing radioactivity in the 31S region or larger were pooled and mixed with deproteinized, uniformly 14C-labeled Ad2 DNA (2 X 104 cpm) purified from virions, and the DNA was precipitated with ethanol. Each DNA sample was digested for 1 hr at 37°C with Hpa I and the resulting fragments were separated by electrophoresis on 1.4% agarose gels (13). The 3H and 13C contents in gel slices corresponding to each of the DNA fragments were measured. The ratio of [3H]DNA (pulse-labeled) to [14C]DNA (uniformly labeled) is expressed as a function of position of each fragment on the genome. The 3H/14C ratio of the G fragment was arbitrarily set as 1, and the other values were normalized to it. The actual cpm in the G fragments were (3H/14C): 1053/321 at 5 min; 1712/379 at 10 min; and 4375/144 at 40 min. The A fragment does not separate from the B fragment under these conditions of electrophoresis. 0-0, 5-min reaction; 0--O, 10-min reaction, A -A, 40-min reaction.
extracts at 370C in the presence of BrdUTP and Ad2 DBP was
analyzed on alkaline CsCl gradients. The density of newly synthesized DNA was approximately 1.8 g/cm3, indicating that it was fully substituted with BrdUMP and not covalently linked to template DNA strands (data not shown). Together, these results indicate that the H5ts125 complemented reaction, which is dependent on exogenous Ad DBP, represents replicative synthesis. DISCUSSION We have detected two patterns of dNMP incorporation catalyzed by souble nuclear extracts of Ad-infected HeLa cells. Synthesis having the characteristics of viral DNA replication required activities that were extractable at high ionic strengths. Considerable dNMP incorporation was found in low ionic strength nuclear extracts, but these reactions were not specific for the Ad DNA-Pro template and the product consisted of short pieces of DNA, possibly due to a random repair reaction. Similarly, the absolute amount of infected nuclear extract was important; the replication reaction required extract concentrations well in excess of those necessary for optimal incorporation of dNMPs. By varying the amount of DNA-Pro in the reaction mixture we found that the capacity for replication was not dependent on the ratio of nuclear extracts to template but required some critical concentration of extracted components (unpublished observations). These results indicate that the study
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Kaplan et al.
5538
of Ad DNA replication in vitro depends on the ability to distinguish easily between random dNMP incorporation and bona fide DNA replication. Two characteristics that we have found useful for this purpose are the dependence of replication on a template containing the 5'-terminal protein and the requirement for functional Ad DBP. Under conditions of replication, the reaction required the Ad DNA-Pro template and would not work on the same DNA after CHC13/isoamyl alcohol denaturation or removal of the terminal protein. Replication catalyzed by H5ts125-infected nuclear extracts was temperature-sensitive, and these extracts could be inactivated by incubation at 370C. The extracts were complemented to full activity by wild-type Ad DBP. However, DBP similarly isolated from H5ts125 cells grown at the permissive temperature did not complement the temperature-sensitive defect at 370C. In these respects, replication of an exogenous template in this system exhibited the same DBP requirement described (14) for extracts that elongate endogenous replicating intermediates. However, DBP purified from E. coli did not substitute for Ad2 DBP in complementing the defective H5ts125 extracts as it had in the endogenous elongation system (26). The reason for the more stringent requirement for the Ad DBP in the exogenous template-dependent system is unknown. It could reflect a specific requirement for the Ad DBP in initiation of DNA synthesis, or it could reflect a differential requirement for DBP in the replication of type I and type II replicating intermediates. Type I intermediates represent the elongation of nascent DNA strands into duplex regions, thus displacing the opposite parental strand. Type II molecules represent the replication of the displaced single strands with the nascent strands growing into single-stranded regions. In the present system, only type I intermediates have been unambiguously identified by electron microscopy (2). In contrast, both type I and type II molecules are replicated in the endogeneous elongation system.
The replicative nature of the reaction with H5ts125 nuclear complemented by the wild-type Ad DBP was confirmed by finding full-sized products in alkaline and neutral sucrose gradients, a shift of newly synthesized BrdUMP-containing strands to a fully heavy density on alkaline .CsCl gradients, and an orderly pattern of chain elongation, initiating at each end of the linear DNA molecule. Inhibition of synthesis in these extracts by aphidicolin and 2',3'-dideoxythymidine triphosphate, which inhibit Ad DNA synthesis in vivo and in other in vitro replication systems, provides additional evidence that this sytem catalyzes true replication (20, 23, 24). Replication in this system was inhibited by the presence of RNases A and T1. In contrast, elongation of replicating intermediates in the previously described endogenous system was RNase resistant (26). The requirement for an RNase-sensitive component in the present system could reflect a requirement for RNA in the initiation of Ad DNA strands or could result from the need for RNA to protect the DNA template from nuclease attack. Further purification of this replicating system should permit a better evaluation of these observations.
extracts
results during the early phases of this study. We also thank Jayne Maritato for her expert technical assistance. These studies were supported by U.S. Public Health Service Grants CA-11512 and CA-21622 from the National Cancer Institute and American Cancer Society Grants NP89K and VC201. L.M.K. is a medical scientist trainee supported by U.S. Public Health Service Grant 5T32 GM 7288 from the National Institutes of Health. H.A. is a research associate supported by Grant P30-CA-13330 from the National Institutes of Health. M.S.H. is the recipient of an Irma T. Hirschl Trust Career Scientist Award. 1. Challberg, M. D. & Kelly, T. J., Jr. (1979) Proc. Natl. Acad. Sci. USA 76, 655-659. 2. Challberg, M. D. & Kelly, T. J., Jr. J. Mol. Biol., in press. 3. Sumida-Yasumoto, C., Ikeda, J., Benz, E., Marians, K. J., Vicuna, R., Sugrue, S., Zipursky, S. L. & Hurwitz, J. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,311-329. 4. Eisenberg, S., Scott, J. F. & Kornberg, A. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,295-302. 5. Richardson, C. C., Romano, L. J., Kolodner, R., LeClerc, J. E., Tamanoi, F., Engler, M. J., Dean, F. B. & Richardson, D. S. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,427-440. 6. Liu, C. C., Burke, R. L., Hibner, U., Barry, J. & Alberts, B. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,469-487. 7. Ginsberg, H. S. & Young, C. H. S. (1976) Adv. Cancer Res. 23, 91-126. 8. Van der Vliet, P. C. & Levine, A. J. (1972) Nature (London) New Biol. 246, 170-174. 9 Van der Vliet, P. C., Levine, A. J., Ensinger, M. & Ginsberg, H. S. (1975) J. Virol. 15, 348-354. 10. Ensinger, M. & Ginsberg, H. S. (1972) J. Virol. 10, 328-339. 11. Van der Vliet, P. C. & Sussenbach, J. S. (1975) Virology 67, 415-426. 12. Van der Vliet, P. C., Zandberg, J. & Jansz, H. S. (1977) Virology
80,98-110. 13. Kaplan, L. M., Kleinman, R. E. & Horwitz, M. S. (1977) Proc. Natl. Acad. Sci. USA 74,4425-4429. 14. Horwitz, M. S. (1978) Proc. Natl. Acad. Sci. USA 75, 42914295. 15. Maizel, J. V., Jr., White, D. 0. & Scharff, M.D. (1968) Virology 36, 115-125. 16. Horwitz, M. S. (1971) J. Virol. 8,675-683. 17. Sharp, P. A., Moore, C. & Haverty, J. L. (1976) Virology 75, 442-456. 18. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 19. Wu, R. (1970) J. Mol. Biol. 51,501-521.
20. Longiaru, M. L., Ikeda, J., Jarkovsky, Z., Horwitz, S. B. & Horwitz, M. S. (1979) Nucleic Acids Res. 6,3369-3386. 21. Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H. & Mano, Y. (1978) Nature (London) 275,458-460. 22. Wist, E. & Prydz, H. (1979) Nucleic Acids Res. 6, 1583-1590. 23. Abboud, M. M. & Horwitz, M. S. (1979) Nucleic Acids Res. 6, 1025-1039. 24. Van der Vliet, P. C. & Kwant, M. M. 25. 26.
27. 28. 29.
grateful to Mark Challberg and Thomas Kelly for their helpful discussions and for sharing critical reagents and unpublished We
are
(1978) Nature (London) 276,532-534. Kedinger, C., Gissinger, F. & Chambon, P. (1974) Eur. J. Biochem. 44, 421-436. Horwitz, M. S., Kaplan, L. M., Abboud, M., Maritato, J., Chow, L. T. & Broker, T. R. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 769-780. Horwitz, M. S. (1976) J. Virol. 18, 307-315. Weingartner, B., Winnacker, E-L., Tolun, A. & Pettersson, U. (1976) Cell 9,259-268. Flint, S. J., Berget, S. K. M. & Sharp, P. A. (1976) Cell 9,559-
571. 30. Sussenbach, J. S. & Kuijk, M. G. (1977)
Virology 77, 149-157.
Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro (eukaryotic DNA replication/soluble nuclear extracts/DNA binding -protein)
LEE M. KAPLAN, HIROYOSHI ARIGA, JERARD HURWITZ, AND MARSHALL S. HORWITZ Departments of Cell Biology, Microbiology and Immunology, Developmental Biology and Cancer, and Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461
Contributed by Jerard Hurwitz, August 7,1979
ABSTRACT Soluble extracts of adenovirus-infected HeLa cell nuclei support DNA replication on exogenous adenovirus DNA templates. Conditions of synthesis using both wild-type and temperature-sensitive extracts have been defined. Nuclear extracts prepared from cells permissively infected with the adenovirus mutant H5ts125 expressed the temperature-sensitive phenotype and could be inactivated at 370C in vitro. These extracts were completely complemented by the addition of wildtype adenovirus DNA binding protein but not by H5ts125 DNA binding protein. Enhancement by binding protein in the mutant extracts represents replication, as demonstrated by the production of full-sized products and orderly chain elongation originating, as in vivo, at both ends of the linear DNA. Replicative synthesis required the 5'-terminal protein bound covalently to template DNA and could be inhibited by denaturation of this 55,000-dalton protein. Various inhibitors of eukaryotic DNA polymerases, such as aphidicolin and 2',3'-dideoxythymidine triphosphate, inhibited replication of exogenous adenovirus templates in this system as they do in previously reported systems that only elongate endogenous replicating intermediates. Challberg and Kelly (1) have recently shown that nuclear extracts of adenovirus type 5 (Ad5)infected HeLa cells catalyzed the semiconservative replication of exogeneously added Ad DNA. DNA synthesis on a template of AdS DNA covalently bound to the 55,00-dalton 5'-terminal protein (Ad5 DNA-Pro) closely resembled synthesis in vvo; however synthesis on deproteinized Ad5 DNA represented a repair-like reaction (1, 2). This system, which presumably initiates and elongates DNA on exogeneously added template, should permit the fractionation and characterization of components required for viral DNA replication. Studies in prokaryotes have depended on the use of conditional-lethal mutants in genes involved in DNA synthesis and the specific inactivation of these genes in cell extracts (3-6). Although analogous mutants in eukaryotic cells have been difficult to obtain, there are three complementation groups of conditional-lethal DNA-negative Ad mutants (7). The defect of one such AdS mutant, H1tsl25, was identified as the gene coding for a 72,000-dalton polypeptide that binds to singlestranded DNA (8, 9). The mutant is temperature-sensitive for viral DNA replication (10), and the DNA binding protein (DBP), isolated from H5ts125-infected cells at permissive temperature, exhibits a temperature-dependent binding to single-stranded DNA cellulose columns that is different from that of the wild-type protein (9). Studies with whole cells infected with H5ts125 and with nuclei prepared from those cells have suggested that the defect in DNA synthesis is due to a failure of initiation (11, 12). Studies with ammonium sulfate nuclear extracts of such cells, which catalyze only the elongation of replicating intermediates previously initiated in vivo (13),
have shown that funtional DBP is also required for the elongation reaction (14). This communication describes further studies of the replication reaction on added exogenous viral DNA catalyzed by nuclear extracts from infected cells. E. tracts prepared from H5tsI25-infected cells were thermolabile and could be inactivated by preincubation at 370C. Under these conditions, full activity was reconstituted by the addition of wild-type Ad2 DBP which generated a reaction having all the characteristics of true replication. We have confirmed that replicative DNA synthesis required viral DNA bound to the 5'-terminal protein (2). Denaturation of this protein inactivated the template activity of the DNA-protein complex. Thus, dependence of DNA replication in vitro on specific viral-coded and virus-associated proteins suggests that the system will allow further study of these and other requirements for DNA replication in eukaryotic systems.
The publication costs of this article were defrayed in part by page
Abbreviations: Ad5, adenovirus type 5; Ad DNA-Pro, adenovirus DNA covalently bound to a 55,000-dalton protein at the 5'-end of each strand; H5ts125, an AdS temperature-sensitive mutant in viral DNA
MATERIALS AND METHODS Cells and Viruses. The sources of HeLa S3 cells, Ad2, and H5ts125 as well as the growth of cells, the growth and purification of stock virions, and infections for nuclear extracts were as described (14, 15). Preparation and Extraction of Infected Nuclei. Cells infected with Ad2 were grown at 370C; those infected with H5ts125 were grown at 32.50C. Two to 4 hr after infection, hydroxyurea was added (final concentration, 3-10 mM), and infections were continued for a total of 18-22 hr for Ad2 and 40 hr for H5ts125. At these times, cells were washed and nuclei were prepared according to the procedure of Challberg and Kelly (1). Nuclei were frozen in liquid nitrogen and stored at -80'C. Frozen nuclei were thawed at 0WC, various amounts of 5 M NaCl were added, and the nuclei were incubated at 0C as described for each experiment. The extracted nuclei and nuclear debris were then removed by centrifugation for 20 min at 15,000 X g in a Beckman type 40 rotor. Preparation of Labeled Viral DNA, DNA-Pro, and DBP. ["4C]Thymidine-labeled Ad2 virion and uniformly "4C-labeled Ad2 deproteinized DNA were prepared as described (16). Ad2 ["4C]DNA-Pro was purified from virions by the method of Sharp et al. (17) and dialyzed against five changes (200 vol each) of 10 mM Tris-HCI, pH 8.1/1 mM EDTA at 40C. [ 5S]Methionine-labeled DBP was purified from HeLa cells infected with either Ad2 or H5ts125 and the purity of these preparations was determined as described (14). Ad2 DBP used in these experiments was approximately 90% pure, and H5ts125 DBP was approximately 70% pure.
charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
synthesis; DBP, viral-coded DNA binding protein. 5534
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Proc. Natl. Acad. Sci. USA 76 (1979)
5535
formed, it was completely solubilized by DNase I at 100
Synthesis of DNA In Vitro. Infected nuclear extracts were incubated in reaction mixtures (0.1 ml) containing-s0 mM Hepes (pH 7.2), 1 mM dithiothreitol, 7.5 mM MgCI2, 2 mM ATP, 50 MM each dATP, dCTP, and dGTP, 5,uM [3H]dTTP (1 X 104 cpm/pmol), and 0.15-0.2 jig of DNA or DNA-Pro template (450-600 pmol of nucleotides). Purified Ad2 or H5ts125 DBP was added to reactions as indicated. For those H5ts125 reactions in which preincubation was carried out, nuclear extracts were incubated in a mixture (80 Al) containing 62.5 mM Hepes (pH 7.2), 0.63 mM dithiothreitol, 6.25 mM MgCI2, 2.5 mM ATP, and DBP as indicated. After preincubation, DNA synthesis was started by the addition of 20 ,u of a solution containing dATP, dCTP, and dGTP each at 250,M, 15 ,M [3H]dTTP, and 450 pmol of Ad2 DNA-Pro. The temperature of preincubations and DNA synthesis reactions was 30 or 370C, as indicated. After incubation, acid-insoluble radioactivity was quantitated as described (13). For nuclease analysis of the product, reactions were stopped by the addition of 1 M NaCI and diluted 1:10 with 20 mM Tris-HCI, pH 7.5/1 mM dithiothreitol/7.5 mM MgGI2; enzymes were added, and the samples were incubated for 30 min at 37°C. Materials. Protein concentrations were determined by the method of Bradford (18) using stock solutions of the proteinbinding dye reagent purchased from Bio-Rad. Hydroxyurea was obtained from either Squibb or Aldrich. All other reagents were as described (13, 14). RESULTS DNA Synthesis Catalyzed by Nuclear Extracts. Nuclear extracts prepared by treating Ad2-infected nuclei with 200 mM NaCl for 60 min were analyzed for their ability to catalyze Ad2 DNA-Pro-directed DNA synthesis (Table 1). The requirements for synthesis in vitro were as described (1). Maximal incorporation of dTMP, using 600 pmol (0.2 Mg) of DNA-Pro template and 25 Ml of extracts (120 Mig of protein), was 7.2 pmol in 1 hr. DNA synthesis required all four dNTPs and Mg+2 in addition to Ad2 DNA-Pro and was stimulated 4-fold by the addition of ATP. Inclusion of all four rNTPs had little effect on the reaction. The reaction was inhibited by the addition of DNase I, a mixture of RNase A and RNase T1, or proteinase K. There was no inhibition by RNase H, and only partial inhibition by the single-strand-specific Neurospora nuclease. Once the product
ug/ml) plus RNase Ti (20 units/ml). Effect of Various Polymerase Inhibitors on Ad2 DNA Synthesis In Vitro. We examined the effect of several inhibitors on DNA replication in the reaction catalyzed by Ad2 nuclear extracts (Table 2). Aphidicolin, which has been shown to inhibit Ad DNA replication in vio and specifically to inhibit DNA polymerase acby 95% at 100,uM (20-22), inhibited replication catalyzed by nuclear extracts. 2',,3'-Dideoxythymidine triphosphate also inhibited synthesis in vitro under conditions at which DNA polymerase y is inhibited but DNA polymerase is completely unaffected (23,24). Actinomycin D inhibited the reaction >90% at 10,Mg/ml. a-Amanitin, a specific inhibitor of RNA polymerase II, had no effect on this reaction, even at concentrations higher than those which will completely inhibit mRNA synthesis (25). Differentiation Between Replicative and Nonreplicative DNA Synthesis In Vitro. We examined the activity of Ad2 nuclear extracts on both Ad2 DNA-Pro and Ad2 DNA templates as a function of the NaCI concentration used to make the extracts (Fig. 1A). Low-salt (0-50 mM) extracts catalyzed similar dTMP incorporation with Ad2 DNA-Pro or DNA but the size of both products on alkaline sucrose gradients was small, suggesting nonreplicative synthesis. High-salt extracts showed a greater discrimination between the two templates and the discrimination with 200 mM extracts was nearly complete. Activity of the Ad2 nuclear extracts was completely dependent on exogenous DNA templates. The product synthesized by 25 ul of the 200 mM Ad2 wild-type nuclear extracts on Ad2 DNA-Pro was full-sized on alkaline and neutral sucrose gradients, and replication originated at both ends of the linear DNA template (data not shown). Therefore, synthesis of fulllength DNA and, particularly, discrimination between the Ad DNA-Pro and Ad DNA templates depended on factors that were extracted in sufficient quantity only by treating nuclei with relatively high (100-200 mM) concentrations of NaCl. With 200 mM nuclear extracts, replicative synthesis required a concentration of extracts greater than that needed for maximal dTMP incorporation (Fig. 1B). Reaction mixtures containing 5 ,l of extracts (25 MAg of protein) incorporated dTMP equally well with Ad2 DNA-Pro and Ad2 DNA templates.
Table 1. Requirements for Ad2 DNA synthesis catalyzed by Ad2 nuclear extracts dTMP incorporated, Conditions pmol/15 min 2.36 Complete - Ad2 DNA-Pro 0.11 - MgCl2 0.08
Table 2. Effect of inhibitors on Ad2 DNA synthesis catalyzed by nuclear extracts dTMP incorporated, % activity Additions pmol/15 min 1.74 None 100 Aphidicolin 10MM 1.32 76
-ATP
0.57
0.29 dATP, dCTP, dGTP + CTP, GTP, UTP (50,uM) 2.60 + DNase I: 50,ug/ml 0.63 100 ug/ml 0.10 + Neurospora nuclease (0.3 unit/ml) 1.85 + RNase A (50,ug/ml), RNase T1 0.17 (10 units/ml) + RNase H (10 units/ml) 2.61 The complete reaction incubated at 370C was as described in Materials and Methods except that the unlabeled dNTPs used were treated with NaIO4 and repurified by DEAE-Sephadex chromatography to remove trace contaminants of ribonucleotides (19). Reaction mixtures contained 600 pmol of Ad2 DNA-Pro template and 25 ,l of nuclear extracts (120 ,g of protein). -
was
,ag/ml but was then unaffected by treatment with RNase A (100
a
100MOM 2',3'-Dideoxythymidine triphosphate 5,MM 15MAM Actinomycin D 1 g/iml 10Mug/im
0.21
12
0.26 0.15
15 9
0.84 0.13
48 7
a-Amanitin
20Mug/ml 50Mug/ml
1.75 101 1.36 78 DNA synthesis reactions were as described in Table 1. Actinomycin D was dissolved in ethanol; aphidicolin was dissolved in dimethyl sulfoxide. These organic solvents were present in the reaction mixtures at concentrations of 1%, which did not affect dTMP incorpo-
ration.
5536
Biochemistry: Kaplan et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
-Q~~~~3-4
,\ fo
03
33
2-
0
1 00 200 NaCI, mM
5
15
Extract, AI
25
X 25
15
5
Extract, Ail
FIG. 1. Differentiation between replicative and nonreplicative dNMP incorporation. (A) Effect of NaCl concentration on extraction of infected nuclei. Ad2-Infected nuclei were prepared and thawed at 00C, aliquots were extracted for 60 min with various concentrations of NaCl (0-200 mM), and activity was measured. Incorporation of [3H]dTMP into acid-precipitable material was measured by using 600 pmol (0.2 pg) of either Ad2 DNA-Pro (-*0) or Ad2 DNA (o -- o) as template in the presence of 25 sl of nuclear extracts (100-120 ,.g of protein). Reactions were at 371C for 30 min. (B) Effect of concentration of nuclear extract. The activity of various concentrations of Ad2-infected nuclear extracts prepared with 200 mM NaCl was measured on Ad2 DNA-Pro (0-0) or Ad2 DNA (O -0). Conditions for extraction and reaction were as in A. (C) Temperature sensitivity of H5ts125 nuclear extracts. H5ts125-infected nuclei were extracted with 100 mM NaCl for 5 min. Activity in various concentrations of extracts was measured for 60 min at either 300C (A-A) or 370C (AA) after a 60-min preincubation at the same temperature; 25 sAl of nuclear extract contained 120 ug of protein. -
---
- -
from cells infected with either Ad2 at 370C or HSts125 at 32.50C (permissive temperature). Various amounts of each DBP were added to the H5ts125 nuclear extracts, the mixtures were preincubated for 60 min at 370C, and DNA synthesis was then assayed (Fig. 2). Purified Ad2 DBP complemented the temperature-sensitive defect, yielding 133% of wild-type activity with 3.2 ,ug of DBP. In contrast, similar concentrations of temperature-sensitive DBP purified from H5ts125 infected cells were unable to complement the defect at 370C. Mixtures of Ad2 DBP and H5ts125 DBP complemented at the level of wild-type DBP used, indicating that the inability of the mutant DBP to complement was not due to an inhibitor in the preparation. Thus, functional DBP is required for DNA synthesis catalyzed by these nuclear extracts. DBP purified from Escherichia colh was previously shown (26) to substitute partially for Ad2 DBP in the DNA elongation reaction on endogenous replicating intermediates. In contrast, E. cohi DBP was unable to substitute for Ad DBP in these extracts which required exogenous DNA. Characterization of the DNA Synthesis Reaction Products. The size and amount of DNA synthesized by H5ts125 nuclear extracts at 300C (permissive temperature), at 370C (nonpermissive temperature), and at 370C in the presence of Ad2 DBP were measured. After a 60-min preincubation and a 60-min reaction at 300C, nearly all of the DNA product sedimented as full-sized Ad DNA in both alkaline (Fig. 3A) and neutral (Fig. 3B) sucrose gradients. When the preincubation and the DNA synthesis reaction were at 370C, dTMP incorporation decreased significantly, although the residual product also sedimented as full-sized viral DNA. Most importantly, when the 370C preincubation and reaction were carried out in the presence of Ad2 DBP, full-length products were formed with
However, the products of reactions with S or 10 ,l of nuclear
extracts on alkaline sucrose gradients were small. Increasing the amount of nuclear extracts to 25 ,l (120 Mg of protein) re-
sulted in the production of full-sized Ad DNA and concurrently generated 20- to 50-fold discrimination between the two templates. Therefore, the DNA replication catalyzed by these extracts was distinct from a repair-type dTMP incorporation and required the presence of Ad DNA covalently bound to the 5'-terminal protein. Ad2 DNA-Pro was inactivated more than 85% by extraction three times with chloroform/isoamyl alcohol, 24:1 (vol/vol). Heating the Ad2 DNA-Pro to 70° in 10 mM Tris-HCl, pH 7.5/1 mM EDTA/10 mM NaCl for 15 or 60 min decreased the activity by approximately 55%. Mixing experiments showed that there were no inhibitory factors in any of the inactive DNA preparations described, and none of these results could be reproduced simply by varying the amount of NaCl in the reaction. Discrimination between replicative and nonreplicative DNA synthesis was also correlated with the thermolability of H5ts125 nuclear extracts. At low concentrations of extract (5 Ml), there was little inactivation of incorporation on Ad2 DNA-Pro templates at 370C (Fig. IC). Under these conditions, the products of the reaction at both 30 and 370C were small on alkaline sucrose gradients. However, with 25 Mul (120 Mug) of nuclear extracts, synthesis was inhibited more than 90% at 370C compared to controls incubated at 300C. At this concentration of extracts, synthesis at 30'C was replicative, as described below. These data suggest that, in addition to decreasing nonspecific incorporation on deproteinized DNA, higher amounts of extracts provided additional activities essential for replicative synthesis. Complementation of the H5ts125 Defect in DNA Replication by Ad2 DBP. Purified wild-type Ad DBP restored the activity of H5ts125 nuclear extracts at 370C. DBP was purified
8
a
'56 E
0
Q., C
2Ho
0
1
2
3
DBP, mg of FIG. 2. Complementation H5ts125-infected nuclear extracts by various DBPs. Various amounts of DBP purified from HeLa cells infected with Ad2 (0-0) or H5ts125 (O- - -0) or from Escherichia coli (A-A) were added to 25 IAI of nuclear extracts prepared from H5ts125-infected cells, and the mixtures were preincubated for 60 min at 370C. Reactions were begun by the addition of [3HldTTP, dATP, dCTP, dGTP, and Ad2 DNA-Pro. H5ts125 nuclear extracts were similarly treated at 300C without the addition of DBP (-). The incorporation of [3HJdTMP into acid-precipiteble material was measured after a 60-min reaction; 25 .l of nuclear extracts contained 120 ,ug of protein.
Biochemistry: Kaplan et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
5537
0
E 10
~0.6
U
3-
5
0.4 0.2
5
10
15 Fraction
5
10
~
-
15
FIG. 3. Sedimentation analysis of DNA synthesized by H5ts125 nuclear extracts in the absence or presence of added Ad2 DBP. The direction of sedimentation in these gradients is from right to left. Aliquots (25 tl) of H5ts125 nuclear extracts containing 120 jug of protein were preincubated for 60 min at 30'C (0-0), at 370C (@--- *), or at 370C in the presence of 1.5 ug of Ad2 DBP (A-A). Reactions were begun by the addition of [3H]dTTP, dATP, dCTP, dGTP, and Ad2 DNA-Pro; incubation was for 120 min at the same temperature as the preincubation. (A) The reactions were terminated by the addition of 0.4 ml of 0.1 M EDTA/0.15 M NaCi at 0C. After removal of 0.1 ml for quantitation of dTMP incorporation, the remaining 0.4 ml was layered onto 5-201% alkaline sucrose gradients and centrifuged at 24,000 rpm in a Beckman SW 27.1 rotor for 16 hr at 4VC. The gradient containing the 301C reaction also contained 5 X 103 cpm of Ad2 [14CJDNA, added as virion before centrifugation. The arrow represents the position of the 34S marker Ad [14C]DNA single strands. Recoveries of 3H were 98% for the 300C reaction, 80% for the 370C reaction, and 93% for the 370C reaction with Ad2 DBP. The pellets of these gradients contained 51, 129, and 79 cpm, respectively. (B) The reactions were stopped with 0.4 ml of 0.5% sodium dodecyl sulfate/10 mM EDTA/50 mM NaCl/10 mM Tris.HCl, pH 7.4. Aliquots (0.4 ml) were layered onto 5-20% neutral sucrose gradients containing 0.5% sodium dodecyl sulfate and centrifuged at 14,000 rpm in a Beckman SW 27.1 rotor for 16 hr at 25°C. The gradient containing the 30°C reaction also contained 5 X 103 cpm of Ad2 [14CJDNA, added before centrifugation; the arrow represents the position of this 31S marker. Recoveries of 3H were 85% for the 30°C reaction, 75% for the 37°C reaction, and 92% for the 37°C reaction with Ad2 DBP. The pellets of these gradients contained 70, 87, and 134 cpm, respectively.
identical sedimentation characteristics as those formed at 300C. Studies with intact cells have shown that Ad DNA replication occurs from both ends of the linear duplex molecules (27-30). Replication in vttro also begins at or near either end of the template as demonstrated by the observation that the terminal restriction fragments of the product are preferentially labeled at early times in the course of this reaction. Only after longer reactions is the distribution of label across the genome more uniform (2). We have confirmed these observations with 200 mM NaCl nuclear extracts of Ad2-infected cells at 370C. To characterize the DNA products synthesized by H5ts125 extracts complemented by Ad2 DBP at 37'C, we used a similar analysis. After preincubation of H5ts125 nuclear extracts in the presence of Ad2 DBP for 60 min at 37°C, DNA synthesis was initiated by the addition of Ad2 DNA-Pro and the four dNTPs. Aliquots were removed at intervals for the subsequent 40 min, and the DNA was purified and digested with Hpa I. The DNA reaction product formed during the first 5 min of incubation was labeled primarily in the terminal fragments (Fig. 4). With increasing time, more label was detected in internal fragments and, after 40 min, the distribution of label across the genome was essentially uniform. Ad DNA synthesized by the H5ts125 nuclear
O
E
C
F
A
B
D G
FIG. 4. Kinetics of labeling of various regions of DNA synthesized by H5ts125 nuclear extracts in the presence of added Ad2 DBP. H5ts125 nuclear extracts (0.25 ml) were preincubated for 60 min at 37°C in the presence of 8 ,ug of Ad2 DBP; the reactions were begun by addition of [3H]dTTP, dATP, dCTP, dGTP, and Ad2 DNA-Pro. Reactions were stopped after 5, 10, or 40 min by the addition of 20 mM EDTA at 0°C. Each aliquot was digested with proteinase K (500 wu/ml) for 30 min at 37°C. The samples were centrifuged in 5-20%o sucrose gradients (11 ml) in 0.5 M NaCl/20 mM Tris.HC1, pH 7.4/10 mM EDTA at 38,000 rpm (Beckman SW 41 rotor) for 2.5 hr at 15°C. All fractions containing radioactivity in the 31S region or larger were pooled and mixed with deproteinized, uniformly 14C-labeled Ad2 DNA (2 X 104 cpm) purified from virions, and the DNA was precipitated with ethanol. Each DNA sample was digested for 1 hr at 37°C with Hpa I and the resulting fragments were separated by electrophoresis on 1.4% agarose gels (13). The 3H and 13C contents in gel slices corresponding to each of the DNA fragments were measured. The ratio of [3H]DNA (pulse-labeled) to [14C]DNA (uniformly labeled) is expressed as a function of position of each fragment on the genome. The 3H/14C ratio of the G fragment was arbitrarily set as 1, and the other values were normalized to it. The actual cpm in the G fragments were (3H/14C): 1053/321 at 5 min; 1712/379 at 10 min; and 4375/144 at 40 min. The A fragment does not separate from the B fragment under these conditions of electrophoresis. 0-0, 5-min reaction; 0--O, 10-min reaction, A -A, 40-min reaction.
extracts at 370C in the presence of BrdUTP and Ad2 DBP was
analyzed on alkaline CsCl gradients. The density of newly synthesized DNA was approximately 1.8 g/cm3, indicating that it was fully substituted with BrdUMP and not covalently linked to template DNA strands (data not shown). Together, these results indicate that the H5ts125 complemented reaction, which is dependent on exogenous Ad DBP, represents replicative synthesis. DISCUSSION We have detected two patterns of dNMP incorporation catalyzed by souble nuclear extracts of Ad-infected HeLa cells. Synthesis having the characteristics of viral DNA replication required activities that were extractable at high ionic strengths. Considerable dNMP incorporation was found in low ionic strength nuclear extracts, but these reactions were not specific for the Ad DNA-Pro template and the product consisted of short pieces of DNA, possibly due to a random repair reaction. Similarly, the absolute amount of infected nuclear extract was important; the replication reaction required extract concentrations well in excess of those necessary for optimal incorporation of dNMPs. By varying the amount of DNA-Pro in the reaction mixture we found that the capacity for replication was not dependent on the ratio of nuclear extracts to template but required some critical concentration of extracted components (unpublished observations). These results indicate that the study
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Kaplan et al.
5538
of Ad DNA replication in vitro depends on the ability to distinguish easily between random dNMP incorporation and bona fide DNA replication. Two characteristics that we have found useful for this purpose are the dependence of replication on a template containing the 5'-terminal protein and the requirement for functional Ad DBP. Under conditions of replication, the reaction required the Ad DNA-Pro template and would not work on the same DNA after CHC13/isoamyl alcohol denaturation or removal of the terminal protein. Replication catalyzed by H5ts125-infected nuclear extracts was temperature-sensitive, and these extracts could be inactivated by incubation at 370C. The extracts were complemented to full activity by wild-type Ad DBP. However, DBP similarly isolated from H5ts125 cells grown at the permissive temperature did not complement the temperature-sensitive defect at 370C. In these respects, replication of an exogenous template in this system exhibited the same DBP requirement described (14) for extracts that elongate endogenous replicating intermediates. However, DBP purified from E. coli did not substitute for Ad2 DBP in complementing the defective H5ts125 extracts as it had in the endogenous elongation system (26). The reason for the more stringent requirement for the Ad DBP in the exogenous template-dependent system is unknown. It could reflect a specific requirement for the Ad DBP in initiation of DNA synthesis, or it could reflect a differential requirement for DBP in the replication of type I and type II replicating intermediates. Type I intermediates represent the elongation of nascent DNA strands into duplex regions, thus displacing the opposite parental strand. Type II molecules represent the replication of the displaced single strands with the nascent strands growing into single-stranded regions. In the present system, only type I intermediates have been unambiguously identified by electron microscopy (2). In contrast, both type I and type II molecules are replicated in the endogeneous elongation system.
The replicative nature of the reaction with H5ts125 nuclear complemented by the wild-type Ad DBP was confirmed by finding full-sized products in alkaline and neutral sucrose gradients, a shift of newly synthesized BrdUMP-containing strands to a fully heavy density on alkaline .CsCl gradients, and an orderly pattern of chain elongation, initiating at each end of the linear DNA molecule. Inhibition of synthesis in these extracts by aphidicolin and 2',3'-dideoxythymidine triphosphate, which inhibit Ad DNA synthesis in vivo and in other in vitro replication systems, provides additional evidence that this sytem catalyzes true replication (20, 23, 24). Replication in this system was inhibited by the presence of RNases A and T1. In contrast, elongation of replicating intermediates in the previously described endogenous system was RNase resistant (26). The requirement for an RNase-sensitive component in the present system could reflect a requirement for RNA in the initiation of Ad DNA strands or could result from the need for RNA to protect the DNA template from nuclease attack. Further purification of this replicating system should permit a better evaluation of these observations.
extracts
results during the early phases of this study. We also thank Jayne Maritato for her expert technical assistance. These studies were supported by U.S. Public Health Service Grants CA-11512 and CA-21622 from the National Cancer Institute and American Cancer Society Grants NP89K and VC201. L.M.K. is a medical scientist trainee supported by U.S. Public Health Service Grant 5T32 GM 7288 from the National Institutes of Health. H.A. is a research associate supported by Grant P30-CA-13330 from the National Institutes of Health. M.S.H. is the recipient of an Irma T. Hirschl Trust Career Scientist Award. 1. Challberg, M. D. & Kelly, T. J., Jr. (1979) Proc. Natl. Acad. Sci. USA 76, 655-659. 2. Challberg, M. D. & Kelly, T. J., Jr. J. Mol. Biol., in press. 3. Sumida-Yasumoto, C., Ikeda, J., Benz, E., Marians, K. J., Vicuna, R., Sugrue, S., Zipursky, S. L. & Hurwitz, J. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,311-329. 4. Eisenberg, S., Scott, J. F. & Kornberg, A. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,295-302. 5. Richardson, C. C., Romano, L. J., Kolodner, R., LeClerc, J. E., Tamanoi, F., Engler, M. J., Dean, F. B. & Richardson, D. S. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,427-440. 6. Liu, C. C., Burke, R. L., Hibner, U., Barry, J. & Alberts, B. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,469-487. 7. Ginsberg, H. S. & Young, C. H. S. (1976) Adv. Cancer Res. 23, 91-126. 8. Van der Vliet, P. C. & Levine, A. J. (1972) Nature (London) New Biol. 246, 170-174. 9 Van der Vliet, P. C., Levine, A. J., Ensinger, M. & Ginsberg, H. S. (1975) J. Virol. 15, 348-354. 10. Ensinger, M. & Ginsberg, H. S. (1972) J. Virol. 10, 328-339. 11. Van der Vliet, P. C. & Sussenbach, J. S. (1975) Virology 67, 415-426. 12. Van der Vliet, P. C., Zandberg, J. & Jansz, H. S. (1977) Virology
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