Theconcentration of2',3'-dideoxythym- idine-5'-triphosphate (ddTTP) was the same as that of dTJP; aphidicolin (gift ofA. H. Todd, Imperial Chemical Industries,.
Proc. NatL Acad. Sci. USA Vol. 78, No. 10, pp. 6206-6210, October 1981 Biochemistry
Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase y (in vitro DNA synthesis/eukaryotic DNA replication/parental replicative form/adenovirus)
REGINE KOLLEK* AND MEHRAN GOULLAN Departnent of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093
Communicated by E. Peter Geiduschek, July 21, 1981
ABSTRACT The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase y.
0.1 M KCV50 mM Tris-HCI, pH 7.5) (14). Assay 2, which detects pol a, pol ,, and pol y, used activated DNA (0.4 mg/ml)/ 50 ,M dATP/50 AM dCTP/10 AuM dGTP/10 puM dlTP/5 mM Mg2/0. 1 M KCV50 mM Tris-HCl, pH 8.5. Assay 3, for optimal detection of pol a activity, used the same mixture as assay 2 except for omission of KCl. Assay 4a, which measured complementary strand synthesis with H-1 viral ssDNA, used reaction mixtures containing viral ssDNA (5 .g/ml) in 50 mM Na N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.6/2 mM potassium phosphate buffer (KPO4), pH 7.6/10 mM KCV 20 mM Na3 phosphoenolpyruvate/5 mM ATP/10 mM MgCl2/ 1 mM ethylene glycol bis(3-aminoethyl ether)-N,N,N',N'-te-
H-1 is an autonomous (helper-independent) member ofthe parvovirus group (1, 2). Its genome consists of a linear singlestranded (ss) DNA (==5 kilobases) with stable hairpin structures at both ends. After attachment to a susceptible cell and uptake of viral DNA, infecting viral ss DNA must first be converted to the double-stranded (ds) replicative form (RF) by synthesis of the complementary strand. The RF undergoes replication, probably by a continuous (as opposed to semidiscontinuous) 5' -- 3' displacement mechanism on both strands (1, 3-5), followed by synthesis and packaging of viral ss DNA. Given the small size of the genome and the known coding requirements for viral capsid polypeptides, it is assumed that replication must depend almost entirely on host functions, as is the case for several other prokaryotic and eukaryotic small DNA viruses. We are studying parvoviral DNA replication as a model for eukaryotic DNA replication and describe here studies on the enzymatic requirements for synthesis of parental RF from infecting viral ss DNA. Relatively little is known from in vivo experiments about this initial step in parvoviral DNA replication. The 3'-terminal foldback could provide a base-paired priming point for synthesis of the complementary strand (1, 2, 6-10), and it is assumed that this functions as the initiation site for synthesis of parental RF in vivo. Although evidence for the viral DNA to RF reaction in vivo has been reported (11, 12), this step has been generally difficult to study, in part due to high particle/ infectivity ratios. In the studies described here, extracts of uninfected cells were analyzed for an activity capable of carrying out the conversion of viral ss DNA to RF DNA. An activity detected in crude extracts was identified as DNA polymerase (pol) y.
traacetic acid/0.6% Nonidet P-40/50 .uM dATP/10 AM dGTP and either 50 AuM dCTP and 10 ,uM [3H]dTI7P (3-6 mCi/ Amol; 1 Ci = 3.7 X 1010 becquerels) or 50 AM dTiP and 10 AM [a-32P]dCTP (10-20 mCi/Amol). In an alternative procedure, assay 4b, ATP, and phosphoenolpyruvate were replaced by 40 mM NaOAc and MgCl2 was reduced from 10 mM to 7 mM. All assay mixtures also contained 1 mM dithiothreitol and bovine plasma albumin (0.2 mg/ml). Results for response to inhibitors represent % activity remaining in the presence of inhibitor compared with 100% in the absence of inhibitor. The concentration of 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) was the same as that of dTJP; aphidicolin (gift of A. H. Todd, Imperial Chemical Industries, London) was at 3 Ag/ml; N-ethylmaleimide (MalNEt) was at 6 mM. All assays were carried out in a total volume of 30 ILI, at 370C, for 30 min (assays 1, 2, and 3) or 60 min (assay 4) (unless stated otherwise). DNA synthesis was determined by incorporation of radioactivity from dNTP into acid-insoluble material. One unit of polymerase activity = 1 nmol of total dNMP incorporated in 60 min at 37C. Fractionation of NB Cell Extracts. Cytosol was prepared from uninfected NB cells by disruption with a Dounce homogenizer followed by centrifugation to remove nuclei. Whole cell extract was prepared by sonification of homogenate (1.6 x 108 cells) in 5 ml of 0.4 M KPO4 (all solutions for this and placental enzyme purification were at pH 7.6 and contained 0.5 mM dithiothreitol) followed by centrifugation (13,000 x g, 30 min) and passage through a column of DEAE-cellulose (4 ml) in the same buffer to remove nucleic acid. The nonadsorbed material was dialyzed against 0.02 M KPO4 and applied to a second DEAE-cellulose column (4 ml), which was eluted with 4.5-ml portions of 0.02, 0.05, 0.1, 0.15, 0.2, and 0.4 M
MATERIALS AND METHODS Cells, Virus, and DNA. Growth of NB cells (human embryonic kidney transformed with simian virus 40), infection with H-1 virus, and preparation of RF DNA have been described (13). Virus grown in NB cells was purified, and the viral DNA extracted, self-hybridized, and purified by gel electrophoresis by procedures that will be described elsewhere. Enzyme Assays. Assay 1, which detects pol y (and pol A), lM dTTP/0.5mM Mn2/ used0.5mM poly(rA).oligo(dl) 1218/10
Abbreviations: ddTTP, 2',3'-dideoxythymidine-5'-triphosphate; ds, double-stranded; KPO4, potassium phosphate buffer; MalNEt, Nethylmaleimide; pol, polymerase; RF, replicative form; ss, singlestranded. * Present address: Heinrich-Pette Institut fir Experimentelle Virologie und Immunologie an der UniversitAt Hamburg, Martinistrasse 52, D2000 Hamburg 20, Germany.
The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. ยง1734 solely to indicate this fact. 6206
~ ~.
Kolleck and Goulian BlProc. Nati Biochemistry:
KPO4. The fractions were dialyzed against 0.04 M KPOd20% ethylene glycol and applied to DNA-cellulose columns (1 ml),
which were eluted with 1.5-ml portions ofthe same buffer containing 0, 0.2, 0.5, and 1.0 M KC1. Fractionation of Extracts from Human Placenta. The initial purification steps were modified from a procedure of Krauss and Linn "15). Tissue (650 g) from term placentas was homogenized in a blender and sonified in an equal volume of 0.4 M KPO4, and the sonicate was centrifuged and passed through a DEAEcellulose column (A) (650 ml) as described above for NB cells. It was then concentrated by precipitation with ammonium sulfate (403 mg/ml) and suspended in 0.02 M KPO4 (300 ml), dialyzed against 0.02 M KPO4, and applied to a second DEAEcellulose column (B) (1.0 liter). The column was washed with 1.5 liters of 0.02 M KPO4, and then eluted with a linear gradient (4 liters) of 0. 02-0.4 M KPO4. By using assays 1, 2, and 3, three different pol activities were distinguished: pol 3, which was not adsorbed to DEAE-cellulose; pol y, which eluted at 0.07 M KPO4 and was active on poly(rA)-oligo(dT); and pol a, eluting at 0.17 M KPO4 and most active with assay 3. The fractions active on H-i DNA, which corresponded to pol y activity, were dialyzed against 0.05 M KPOV20% ethylene glycol and applied to a column of phosphocelluloset (120 ml), which was washed with 240 ml of the same buffer and eluted with 500 ml of a linear gradient of 0.05-0.4 M KPOd20% ethylene glycol. The fractions containing the ability to replicate H1 DNA (assay 4b) and poly(rA)-oligo(dT) (assay 1) (peak at 0.2 M KPO4) were dialyzed against 0.04 M KPOd20% ethylene glycol and applied to a column of DNA-cellulose (6 ml), which was washed with 9 ml of the same buffer and then with a linear gradient (25 ml) of 0-0.5 M KC1 in the same buffer (residual protein on the column removed with 1.0 M KC1 contained no polymerase activity). Pol y eluted at 0.38 M KC1 in a single peak that contained the activity on H-1 DNA. Traces of pol a activity eluted at 0.1 M KCl, well separated from pol y. Enzyme fractions were concentrated by dialysis against solid sucrose, dialyzed against 40 mM KPOd30% ethylene glycol, and stored at -200C or -700C. A portion (1 unit, assay 2) of the DNA-cellulose fraction of pol y was purified further by centrifugation in a glycerol gradient (10-30%) in 20 mM KPOJ0.5 M KCV/0.1 mM EDTA containing bovine plasma albumin (0.25 mg/ml) for 13 hr at 50,000 rpm in a Beckman SW-60 Ti rotor at 10C. Markers of aldolase (Mr 158,000) and bovine plasma albumin (Mr 68,000) were in a separate tube run in parallel. Pol y and the activity on H-1 DNA appeared in the same fractions. Gel Electrophoresis. Samples were treated with Pronase (2 mg/ml) in the presence of 2% NaDodSO4 at 370C for 16 hr and then extracted with phenol and precipitated with ethanol. Gel electrophoresis was carried out in 1% agarose/40 mM Tris base/ 20 mM acetic acid/2 mM EDTA containing ethidium bromide (0.5 ttg/ml). For radioautography, gels were dried and exposed to Kodak XR-5 film. RESULTS Activity of Cell Extracts on H-1 Viral DNA. To detect the activity that converts viral ss DNA to RF, whole cytosol from uninfected NB cells was incubated with viral DNA. The conditions used were based on those found optimal for synthesis of viral RF DNA in nuclei from infected cells (unpublished results). The product that resulted included a small proportion of molecules that reached the full size of ds RF DNA (Fig. 1, t In some preparations, particularly those starting with placentas from the second trimester, in which the specific activities of the DEAE-cellulose fraction and preceding steps were higher, the phosphocellulose step was omitted without obvious effect on the final result.
0-
-
-
-
RF-
a
b
Acad. Sci. USA 78 (1981)
-.-
c
d
6207
:-
e
f
g
FIG. 1. Reaction of H-1 viral ss DNA with cytosol of human NB cells. Viral DNA was incubated (assay 4a; [a-32P]dCTP) with cytosol (equivalent to 6 x 105 cells), and the DNA was analyzed by agarose gel electrophoresis followed by radioautography. All incubation mixtures were complete except for a. Lanes [addition (if any), incubation period, total incorporation]: a, no viral DNA, 30 min, 0.3 pmol; b, 10 min, 4.5 pmol; c, 30 min, 10.7 pmol; d, 60 min, 15.7 pmol; e, ddTTP, 30 min, 5.7 pmol; f, aphidicolin, 30 min, 5.1 pmol; g, MalNEt, 30 min, 0.9 pmol. 0, origin; V, ss viral DNA; RF, ds RF DNA.
lanes b-d). With longer incubation periods, the amount of RF increased but, in addition, the proportion oflabel that migrated faster than viral ss DNA also increased, reflecting nuclease activity in the extract. The synthesis required the ss DNA as template (Fig. 1, lane a), but the ss DNA was rapidly degraded; only traces remained after 10 min, and none was detected after 30 min except in- the presence of MalNEt (not shown). (The efficiency of conversion to RF was greater after storage of extracts, apparently because of reduced nuclease activity.) Both aphidicolin, which specifically inhibits pol a, and dd1TP, which inhibits pol y > pol ,/ (but not pol a under these conditions), reduced synthesis by about one-half. The size distribution of the product synthesized in the presence or absence of aphidicolin did not appear to be different; however, ddTIP substantially reduced the proportion of longer molecules in the product (Fig. 1, lanes e and f). MalNEt, which inhibits pol a and pol y but not pol /3, strongly inhibited synthesis; the small amount of residual product migrated with viral ss DNA template on gels (Fig. 1, lane g). Further attempts to characterize the activity(s) responsible for synthesis of the complementary strand on the viral DNA template followed the route of purification. Partial Purification from NB Cell Extracts. A crude extract prepared from a homogenate of NB cells was fractionated on a DEAE-cellulose column. Maximum synthesis with H-1 DNA template was found in the fraction (0.1 M KPO4) that contained the highest activity with poly(rA)-oligo(dT) (Fig. 2), which indicates that it contained most ofthe pol y (14). Smaller amounts of activity on H-1 DNA were present in the other fractions. DNA polymerase activities were further identified by the use of inhibitors. The activity on poly(rA) oligo(dT) (assay 1) in the
Biochemistry: Kollek and Goulian
6208
Proc. Nad Acad. Sci. USA 78 (1981)
00-
a
b
c
d
f
e
h
9
0 0
E RF:4..w ._
C 0
m
0 C
non
adsorb.
0.05
0.15.
0.1 M
KPO4
0.2
0.4
V-_ I
FIG. 2. Fractionation of NB whole cell extract by DEAE-cellulose chromatography. After removal of nucleic acids (DEAE-cellulose column A), NB whole cell extract was applied to a second DEAE-cellulose column (B) and eluted at various concentrations of KPO4. Nonadsorb, fraction that did not adsorb when the sample was applied to the column. Activity in fractions was determined by assays 3 (A), 1 (B), and 4b (30-min incubation) (C). Total activity of the extract (units) before application to DEAE-cellulose column B and total recovery (%) from DEAE-cellulose column B (including the nonadsorbed fraction) were 423 units and 81%; 85 units and 79%; and 23 units and 59% in A, B, and C, respectively.
0.1 M KPO4 fraction was strongly inhibited both by ddTTP (4%) and by MalNEt (6%), confirming its identity as pol y. pol identified by partial sensitivity to ddl7P (72%) and resistance to aphidicolin (100%) and MalNEt (88%), was, as expected (16), not adsorbed by DEAE-cellulose (assay 2). The predominant polymerase activity of the extract eluted primarily in the 0.15 and 0.2 M KPO4 fractions (assay 3) and was resistant to ddTTP (89% and 100%, respectively) but sensitive to aphidicolin (21% and 13%, respectively) and to MalNEt (
