Eur J Biochem 269, 2383–2393 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02902.x Re-oxygenation of hypoxic simian virus 40 (SV40)-infected CV1 cells causes distinct changes of SV40 minichromosome-associated replication proteins Hans-Jorg Riedinger, Maria van Betteraey-Nikoleit and Hans Probst ă Physiologisch-Chemisches Institut der Universitaăt Tuăbingen, Germany Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the origin region After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place To further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase d, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase a, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work DNA replication in mammalian cells is subject to a regulation, which depends on the O2 tension in the cellular environment This regulation results in inhibition of cellular replicon initiation when the concentration of O2 falls below 0.1% Re-oxygenation after several hours of hypoxia causes a burst of new initiations within a few minutes So far, this regulatory phenomenon has been demonstrated for Ehrlichascites, HeLa and CCRF cells [1–3] and it may be a general mechanism, which adapts the cellular DNA replication to the supply of O2 and other nutrients This seems to be of particular significance during embryonic growth, wound healing or tumour cell propagation The mechanism leading from re-oxygenation to replicon initiation is largely obscure The remarkably fast resumption of initiations after re-oxygenation suggests that the O2-dependent replication control acts very directly on the replication apparatus O2-Dependent regulation of replicon initiation was also demonstrated for viral replication in simian virus 40 (SV40)infected CV1 cells [4,5] As the replication of SV40 is relatively well investigated, this virus seems to be well suited to examine the events leading to the reversible shutdown of replicon initiations by hypoxia As we have shown, reduction of the pO2 to 0.1–0.02% suppresses the viral DNA synthesis Re-oxygenation results in new initiations followed by an almost synchronous round of SV40 replication This synchronous round of replication was shown to begin at the viral origin [4] After re-oxygenation, the viral replication starts with the unwinding of the viral origin region [5] This was shown by detection of a highly underwound SV40 topoisomer (form U) about after re-oxygenation Form U was not detectable under hypoxia Primer RNA-DNA synthesis was started  3–5 after re-oxygenation As form U turned out to contain primer RNA-DNA, unwinding and primer synthesis may occur more or less concomitantly after the initial opening of the viral origin region In vitro, the events leading to unwinding of the viral origin and subsequent DNA synthesis are characterized by the binding of replication proteins to the SV40 origin First, in an ATP-dependent reaction, SV40 large T antigen binds as a double hexamer to the viral origin, leading to local distortions of the origin region [6–11] Further local unwinding, catalysed by the helicase activity of the T antigen, depends on the binding of replication protein A (RPA) and topoisomerase I [12–15] After a stretch of Correspondence to H.-J Riedinger, Physiologisch-chemisches Institut der Universitat Tubingen, Hoppe-Seyler-Straòe 4, D-72076 Tubingen, ă ă ă Germany Fax: + 49 7071293339, Tel.: + 49 70712972454, E-mail: hans-joerg.riedinger@uni-tuebingen.de Abbreviations: SV40, simian virus 40; RPA, replication protein A; RFC, replication protein C; PCNA, proliferating cell nuclear antigen; ATM, ataxia telangiectasia-mutated (Received 18 December 2001, revised 20 March 2002, accepted 22 March 2002) Keywords: hypoxia; DNA unwinding; SV40; large T antigen; replicon initiation 2384 H.-J Riedinger et al (Eur J Biochem 269) unwound DNA is generated, DNA polymerase a-primase synthesizes RNA-DNA primers at the single-stranded templates [16–18] Elongation of these primers involves replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase d [19–21] In the present communication, we further try to define the state, at which hypoxia interrupts the initiation of SV40 replication in vivo, by examining which replication proteins are present in the viral minichromosome before and after re-oxygenation The presented data indicate that unwinding occurs immediately after re-oxygenation but not under hypoxia We further demonstrate that a significant fraction of the proteins engaged in viral replication is bound to the SV40 minichromosome already under hypoxia, but that none of the protein complexes necessary for unwinding, primer synthesis and elongation seems to be completed before the respective events actually take place MATERIALS AND METHODS Transient hypoxia, re-oxygenation and radioactive labelling Monkey CV1 cells (ATCC CCL 70) were grown and infected with SV40 as described previously [22] Transient hypoxia was started 36 h after infection by gassing with 0.04% O2/5% CO2 and Ar to 100% for h [4] For re-oxygenation, 0.25 vol of medium equilibrated with 95% O2/5% CO2 was added to the hypoxic cell culture and gassing was continued with artificial air [4] To label newly formed DNA, [methyl-3H]deoxythymidine was either added directly to the cells or, for hypoxic labelling, by plunging a spatula carrying the appropriate quantity in dried form into the culture medium For long-term labelling of DNA, [2-14C]deoxythymidine (5 nCiỈmL)1) was added to the cell cultures immediately after infection with SV40 Staurosporine (Roche, Mannheim, Germany), olomoucine (ICN, Eschewed, Germany) or wortmannin (Alexis, Grunberg, Germany), dissolved in dimethylsulfoxide, were ă applied to hypoxic cell cultures on a spatula after gassing in a hypoxic chamber for 30 To stop incubations, the culture medium was removed by aspiration and the cells were washed twice with ice-cold NaCl/Pi [150 mM NaCl, 10 mM NaHPO4, (pH 7.0)] The determination of acid-insoluble radioactivity has been described previously [23] Preparation of SV40 minichromosomes Preparation of SV40 minichromosomes was performed essentially as described by Su & DePamphilis [24] In brief, SV40-infected CV1 cells of Petri dishes 132 mm in diameter were used for preparation of the minichromosomes Stopped cell cultures were washed twice with ice-cold hypotonic buffer [10 mM Hepes/KOH (pH 7.8), mM KCl, 0.5 mM MgCl2, 0.1 mM dithiothreitol] Cells were homogenized with five strokes in a Dounce homogenizer and the nuclei were pelleted by centrifugation at 3000 g for After resuspension in hypotonic buffer containing protease inhibitor cocktail (Sigma, Deisenhofen, Germany), the nuclei were eluted for 1.5 h at °C and then pelleted by centrifugation at 8000 g for 10 The minichromosomes Ó FEBS 2002 in the supernatant were sedimented at 14 000 g in a Beckman TLA 100.2 rotor for 25 at °C and resuspended in an appropriate buffer Addition of ATP (4 mM) during the preparation of the minichromosomes was never found to change the results obtained This step was therefore generally omitted Alkaline sedimentation analysis of viral DNA in isolated minichromosomes and in nuclei SV40-infected cells were incubated at atmospheric pO2 or re-oxygenated after h of hypoxia for 10 or 25 Ten minutes before the end of incubation, cells were pulselabelled with 10 lCiỈmL)1 [methyl-3H]deoxythymidine Incubation was then stopped and minichromosomes were eluted from the nuclei for 1.5 h as described above After sedimentation of the nuclei, the minichromosome-containing supernatant or the nuclei resuspended in NaCl/Pi were brought to 0.2 M NaOH, incubated for h at room temperature and loaded on top of a 5–40% linear sucrose gradient in 0.25 M NaOH, 0.6 M NaCl, mM EDTA, 0.1% sodium lauroylsarcosinate After centrifugation in a Beckman SW40 rotor at 164 000 g and 23 °C for 16 h, 0.6-mL fractions were collected from the top of the gradient and analysed for acid-insoluble radioactivity [23] Electrophoresis of minichromosome-bound proteins, Western blotting Minichromosomes resuspended in NaCl/Pi were diluted with 10 vol of 10 mM sodium pyrophosphate and 10 mM EDTA (pH 8.0), and proteins were extracted with vol of phenol (pH 8.0) The phenolic phase was then extracted twice with the same volume of 10 mM sodium pyrophosphate, 10 mM EDTA (pH 8.0) and proteins were precipitated by addition of vol of acetone at )20 °C overnight After centrifugation at 200 000 g, °C for 45 min, the pellet was successively washed with chloroform/CHCl3 and methanol, dried and redissolved in mM Tris/HCl (pH 7.5) The proteins were separated on an 8% SDS/polyacrylamide gel [25] and then blotted onto a nitrocellulose membrane with a semidry blot device (Pharmacia, Freiburg, Germany) Immunodetection of replication proteins was done with the ECL Western blotting kit (Amersham, Freiburg, Germany) according to the protocol of the manufacturer Dilutions of antibodies used were as follows: T antigen (monoclonal antibody, clone pAB 101, kind gift of H Stahl, Homburg/Saar, Germany), : 100 of the hybridoma supernatant; RPA (monoclonal antibodies against the 34-kDa and 70-kDa subunits, kind gifts from J Hurwitz, Memorial Sloan Kettering Cancer Center, New York, USA), : 200 and : 100 of the hybridoma supernatant, respectively; topoisomerase I (polyclonal antibody, TopoGen Inc., Columbus, USA), mL)1; primase (polyclonal antibody against the 48-kDa subunit, kind gift of H.-P Nasheuer, Institut fur medizinische Biochemie, ă Jena, Germany), : 2000; polymerase a (polyclonal antibody against the 180-kDa subunit, kind gift of H.-P Nasheuer, Institut fur medizinische Biochemie, Jena, ă Germany), : 1000; RFC (polyclonal antibody against the 37-kDa subunit, a kind gift of J Hurwitz, Memorial Ó FEBS 2002 Initiation of SV40 replication in vivo (Eur J Biochem 269) 2385 Sloan Kettering Cancer Center, New York, USA), : 1000; PCNA (monoclonal antibody, clone 19F4, Roche, Mannheim, Germany) lgỈmL)1; polymerase d (monoclonal antibody against the 125-kDa subunit, Transduction laboratories, Heidelberg, Germany) : 1000 Digestion of immunoprecipitated minichromosomes with micrococcus nuclease and correlation of the digestion products with the SV40 genome SV40-infected cells were incubated hypoxically for h and simultaneously labelled with [methyl-3H]deoxythymidine (10 lCiỈmL)1) Thereafter, cells were either stopped or re-oxygenated for and then stopped Minichromosomes were isolated as described above For immunoprecipitation, 10 mL of T antigen-specific hybridoma supernatant (clone pAB 101) were incubated with protein A agarose (20 mg, Biorad, Richmond, USA) for h at °C Thereafter, protein A agarose was pelleted and minichromosomes were bound by incubation for 90 at °C in NET buffer [150 mM NaCl, 50 mM Tris/HCl (pH 7.5), mM EDTA, 0.5% Nonidet P40] The immunoprecipitate was then washed three times with NET buffer and resuspended in 500 lL Tris/HCl, pH 7.4 (10 mM), CaCl2 (1 mM) Subsequently, digestion with micrococcus nuclease (48 U) was performed at 37 °C for 25 The immunoprecipitate and the supernatant were then incubated for h at 37 °C with proteinase K (40 lgỈmL)1), SDS (0.5%) and extracted with phenol/ CHCl3 The DNA was precipitated with ethanol, redissolved and hybridized against membrane-fixed, singlestranded M13mp18 DNA containing segments of the SV40 genome cloned in either direction into the plasmid SmaI site by standard procedures [26] For fixation of the single-stranded SV40 probes onto nylon membrane, 400 ng DNA was dissolved in 200 lL 10 · NaCl/Cit (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0), heated to 65 °C for 10 and chilled on ice The DNA was then transferred to the membrane (2 cm in diameter), dried at 37 °C and fixed by UV irradiation Hybridization was performed at 37 °C, as described previously [27] After hybridization, the radioactivity bound to each of 14 probes, representing both complementary DNA strands of seven SV40 genome fragments (see inset in Fig 3), was quantified and normalized for size of the respective SV40 segment Nuclease P1 digestion of SV40 minichromosomes SV40 minichromosomes isolated from hypoxic and re-oxygenated cell cultures were digested with nuclease P1 essentially as described by Adachi & Laemmli [28] After sedimentation, the minichromosomes were redissolved in HMN buffer [5 mM Hepes/NaOH (pH 7.5), mM MgCl2, 100 mM NaCl] and one half was digested with U of nuclease P1 [Pharmacia, Freiburg, Germany; lL)1 stock in 8.5 mM sodium acetate (pH 6.0), 50% glycerol] for 10 s at 37 °C Digestion was stopped by the addition of 15 vol of ice-cold RIPA buffer Thereafter, minichromosomes were immunoprecipitated with T antigen-antibody-saturated protein A agarose and digested with proteinase K, as described above After phenol/CHCl3 extraction, DNA was precipitated with ethanol and analysed on a 1% agarose gel in TAE buffer [40 mM Tris, mM sodium acetate, mM EDTA (pH 7.8)] SV40 DNA isolation from cell cultures, chloroquine gel electrophoresis, Southern blotting, hybridization SV40 DNA from whole cells was isolated as described previously [5] Washed cells were lysed and digested in 0.25 M EDTA (pH 8.0), 1% sodium lauroylsarcosinate and 100 lgỈmL)1 proteinase K at 55 °C for h The lysate was then extracted twice with phenol/CHCl3 and dialyzed against mM Tris/0.1 mM EDTA (pH 8.0) at °C overnight After digestion with RNase A (100 lgỈmL)1 at 37 °C for h), 100 ng of isolated DNA per slot was loaded onto a 25 · 20 cm agarose gel containing 20 lgỈmL)1 chloroquine in gel buffer (30 mM NaH2PO4, 36 mM Tris, mM EDTA) Electrophoresis was carried out at VỈcm)1 and °C for 20 h Southern blotting was performed under alkaline conditions [26] The DNA was detected by hybridization [27] using a 32P-labeled, BamHI-linearized SV40 probe Competition of RPA-34 binding to minichromosomes by single-stranded DNA SV40 minichromosomes of hypoxic and re-oxygenated cell cultures were eluted as described above After sedimentation of the nuclei, the supernatant was divided and one half was incubated with 40 lgỈmL)1 sonicated, heat-denatured herring sperm DNA for 30 at 30 °C, whereas the other half was incubated without competitor DNA Thereafter, the minichromosomes were pelleted by ultracentrifugation, resuspended in NaCl/Pi and processed for Western blotting as described above RESULTS Eluted minichromosomes represent the state of viral replication in SV40-infected cells Elution of SV40 minichromosomes from isolated nuclei of infected CV1 cells in hypotonic buffer usually yielded 35% of total viral minichromosomes (data not shown) In order to show that the eluted fraction of SV40 minichromosomes is representative of the overall SV40 replication in the infected cells, SV40 DNA isolated from minichromosomes was compared with that remaining in the eluted nuclei, using alkaline sedimentation analysis SV40-infected CV1 cells were either grown under atmospheric pO2 or re-oxygenated after h hypoxia for 10 or 25 and labelled with [methyl-3H]deoxythymidine during the last 10 of the incubation SV40 minichromosomes and resuspended eluted nuclei were brought to 0.2 M NaOH and incubated for h at room temperature H-Labeled DNA in the lysates was then analysed for size by alkaline sucrose gradient centrifugation As a control, noninfected, normoxically cultivated CV1 cells where treated exactly in the same way Figure shows that there were no significant differences between the peak positions, i.e the lengths of growing DNA strands, in minichromosomes and nuclei Incubating cell cultures under atmospheric pO2 (Fig 1A,B) resulted in peaks around fraction 10, representing full-length SV40 DNA Additionally, a peak at fraction 18, representing 2386 H.-J Riedinger et al (Eur J Biochem 269) Ó FEBS 2002 preferably remain in the nuclei, as only 10–20% were eluted compared to 30–50% of the pulse-labelled (replicating) SV40 DNA (squares) With re-oxygenated cells, peaks at fraction 8, representing growing DNA of  kb, 10 after re-oxygenation (Fig 1C,D), and at fraction 11, representing full length DNA, 25 after re-oxygenation (Fig 1E,F), were obtained Essentially the same profiles were found when viral DNA of whole SV40-infected cells cultivated under the same conditions was analysed [4,5] The fraction of eluted SV40 minichromosomes was therefore taken to be representative of the overall minichromosomes in infected cells Figure 1G,H shows the sedimentation profiles of noninfected CV1 cells As expected, no DNA was found in the eluate of nuclei (Fig 1G), whereas DNA of the nuclei sedimented to the bottom (fraction 20) of the sucrose gradient (Fig 1H) These results suggest that the sedimentation profiles shown in Fig 1A–F are specific for SV40 Differential pattern of minichromosome-bound replication proteins occur before and after re-oxygenation of hypoxic cells Fig Alkaline sucrose gradient centrifugation of viral DNA of minichromosomes eluted from nuclei of normoxically cultivated or re-oxygenated SV40-infected CV1 cells and of the nuclei remaining after the elution Cells were cultivated under normoxic conditions (A,B) or re-oxygenated after h of hypoxia for 10 (C,D) or 25 (E,F) and labelled with 10 lCi of [methyl-3H]deoxythymidine per mL during the last 10 of the incubation (j) Cell nuclei were eluted with hypotonic buffer and the DNA of the minichromosomes (A,C,E) and of the nuclei (B,D,F) was analysed by alkaline sedimentation (G,H) Sedimentation of DNA of noninfected CV1 cells treated exactly as (A,B) Sedimentation was from left to right (d) DNA labelled by addition of [2-14C]deoxythymidine (5 nCiỈmL)1) immediately after infection of CV1 cells with SV40 covalently closed, supercoiled SV40 DNA was visible Accordingly, this peak was predominant in long-term labelled DNA (circles) Minichromosomes containing long-term labelled (supercoiled) SV40 DNA seemed to Initiation of SV40 DNA replication is inhibited at a stage before unwinding in hypoxically cultivated cells After re-oxygenation, unwinding and RNA-DNA primer synthesis are detectable after  [5] We asked whether re-oxygenation is accompanied by sequential binding of different replication proteins, which are necessary to catalyse these steps SV40-infected cell cultures were grown hypoxically for h and stopped or re-oxygenated for 1, 3, or and then stopped Proteins of SV40 minichromosomes were prepared, separated by SDS/PAGE and blotted to a nitrocellulose membrane Chosen proteins were then detected with specific antibodies As quantification of Western blots is known to be difficult, reproducibility of the results was carefully ascertained, especially in cases where changes before and after re-oxygenation were found to be small (e.g for T antigen or polymerase a-180) Each protein was tested in at least three independent experiments and representative results are shown in Fig RPA-34, topoisomerase I, primase-48, RFC-37, and polymerase d-125 were associated with the SV40 minichromosomes already under hypoxia For RPA-34 and RFC-37, longer exposure times were chosen to accentuate slower migrating bands If at all, only minor differences in signal intensities were found for these proteins irrespective whether they were isolated from minichromosomes of hypoxic or re-oxygenated cell cultures In case of RPA-34 and RFC-37, this was confirmed by shorter exposure (data not shown) Topoisomerase I, the 34-kDa subunit of RPA and the 37-kDa subunit of RFC seemed to exist in several modifications (Fig 2) Two dimensional gel electrophoresis (first dimension: isoelectric focusing; second dimension: SDS/ PAGE) of SV40 minichromosome-bound proteins, Western blotting and immunodetection revealed that, for RPA-34 and RFC-37, these modifications differed significantly in their isoelectric points but only slightly in their molecular masses (data not shown) This suggests that they are differently phosphorylated species The patterns of topoisomerase and RFC-37 were the same irrespective of the Ó FEBS 2002 Initiation of SV40 replication in vivo (Eur J Biochem 269) 2387 Fig Immunodetection of minichromosomeassociated replication proteins Minichromosome-bound proteins isolated from hypoxic (H) or re-oxygenated (re-oxygenation times are indicated) SV40-infected cell cultures were separated by SDS-PAA gel electrophoresis, Western blotted and detected with appropriate antibodies incubation conditions, whereas RPA-34 was obviously phosphorylated additionally after re-oxygenation The degree and timing of the appearance of additional phosphorylations of RPA-34 varied somewhat in different experiments (compare Fig with Fig 6B) First changes of the phosphorylation pattern were, however, almost always detectable as early as after re-oxygenation Two bands were detectable for the catalytic subunit of polymerase d As the difference in electrophoretic mobility was rather pronounced considering the molecular mass of polymerase d, it seems unlikely that the two bands represent different phosphorylation forms of the protein Rather, the lower band may be a degradation product of the upper Recently, Schumacher et al [29] isolated a N-terminal truncated form of polymerase d with an apparent molecular mass of 116 kDa, approximately the same size as the lower band of polymerase d detected by us The ratio of the intensities of both bands significantly changed The upper band increased and after re-oxygenation, while the lower band decreased The sum of the bands remained, however, largely constant This was especially evident in preparations that yielded essentially equal signal intensities of both bands from the beginning Supposing the lower band to be a degradation product of the upper, this result could indicate that polymerase d was better protected against degradation when it was stably bound to its target at the replication fork, i.e during primer elongation An unexpected binding behaviour showed T antigen Under hypoxia, it was associated with the chromatin but decreased immediately after re-oxygenation to about one third Three minutes after re-oxygenation, the amount of minichromosome-bound T antigen increased again and attained the level seen under hypoxia until after re-oxygenation The 70-kDa subunit of RPA, the 180-kDa subunit of DNA polymerase a, and PCNA were barely detectable in hypoxic minichromosomes After re-oxygenation, the amount of all three proteins significantly increased, though at different times RPA-70 increased after just and further increased up to min, remaining then at a constant level The amount of polymerase a-180 first increased after re-oxygenation and remained unchanged thereafter PCNA also increased after and further increased until after re-oxygenation These binding patterns suggest that the proteins are recruited to the minichromosome (at least in a form bound stably enough to resist minichromosome isolation) in the same order they actually start to function in replication T antigen is bound to the viral origin of replication in SV40 minichromosomes Figure shows that T antigen is detectable at the SV40 chromatin under hypoxia and after re-oxygenation As several T antigen-binding sites exist in the SV40 genome, we determined the genome region, where T antigen is bound under hypoxia and after re-oxygenation SV40 minichromosomes labelled with [methyl-3H]deoxythymidine were isolated from hypoxic or re-oxygenated cell cultures and immunoprecipitated using protein A agarosebound T antigen antibody The immunoprecipitate was resuspended and digested with micrococcus nuclease After digestion, the immunoprecipitate was pelleted by centrifugation, and DNA fragments were isolated from the pellet and the supernatant and hybridized against membranefixed single-stranded probes containing different segments of the SV40 genome (see inset of Fig 3) For each SV40 restriction fragment, two single-stranded probes, representing both complementary DNA strands, existed The sum of radioactivity bound by both probes of each fragment is shown in Fig DNA isolated from the immunoprecipitate, i.e DNA fragments that were associated with T antigen before purification, mainly hybridized to SV40 probes 1a and 1, irrespective whether cells were re-oxygenated for or kept hypoxic (Fig 3A,C) This means that T antigen is preferably bound to the core origin (represented by probe 1a) or to the whole SV40 origin region (represented by probe 1) After re-oxygenation, but not under hypoxia, some DNA also hybridized to probe 4, indicating that T antigen was also associated with SV40 DNA containing the termination region at this time As of re-oxygenation are not sufficient to allow complete replication of the whole SV40 genome [4,5], association of T antigen with this fragment likely results from elongation of SV40 replicons, which were started, but not yet terminated, before or during hypoxia DNA fragments isolated from the supernatant of the immunoprecipitation hybridized more or less uniformly to all probes except 1a (Fig 3B,D) As 1a represents the SV40 core origin, this result indicates that, in most minichromosomes, the core origin is protected against digestion with micrococcus nuclease by association with T antigen 2388 H.-J Riedinger et al (Eur J Biochem 269) Ó FEBS 2002 Fig Localization of T antigen at the SV40 chromatin 3H[thymidine]-labelled minichromosomes isolated from hypoxic (A,B) or re-oxygenated (C,D) SV40-infected CV1 cells were immunoprecipitated with T antigen antibody-saturated protein A agarose The immunoprecipitate was resuspended and digested with micrococcus nuclease After centrifugation, DNA fragments were isolated from the immunoprecipitate and the supernatant and analysed by hybridization against membrane-fixed single-stranded M13mp18 DNA containing segments of the SV40 genome (see inset) For each SV40 restriction fragment, two single-stranded probes, representing both complementary DNA strands, existed The radioactivity bound to the membrane-fixed SV40 probes was measured, normalized for fragment size, and added up for both probes of each SV40 genome segment (A,C) Membrane-bound DNA fragments of the immunoprecipitate (A) hypoxic cells, (C) cells re-oxygenated for min; (B,D) Membrane-bound DNA fragments of the supernatant (B) hypoxic cells, (D) cells re-oxygenated for Inset: Cleavage sites of restriction enzymes used to generate the set of SV40-specific single-stranded DNA probes Restriction fragment 1a harbours the SV40 core origin of replication Sensitivity of SV40 minichromosomes to single-stranded DNA-specific nuclease P1 increases after re-oxygenation The slight increase in the amount of minichromosomebound RPA-70 together with additional phosphorylation of RPA-34 after re-oxygenation (Fig 2) indicates the occurrence of single-stranded DNA [30–34] and therefore probably the beginning of unwinding of the SV40 chromatin (see below) To independently examine the existence of unwound DNA, we used the single-stranded DNA-specific nuclease P1 This enzyme has been successfully used in a similar study by Adachi & Laemmli to detect unwound DNA regions in Xenopus sperm nuclei [28] SV40 minichromosomes of hypoxic and re-oxygenated cell cultures were divided in two halves, and one half was exposed to nuclease P1 and immunoprecipitated, while the other half was immunoprecipitated without digestion After that, the SV40 DNA was isolated, separated on a 1% agarose gel, blotted and detected by hybridization using a 32 P-labeled SV40-specific probe The signal intensities of supercoiled and open circle SV40 DNA of P1-digested and undigested minichromosomes were quantified densitometrically and compared (the undigested signal was set as 100%) Essentially no difference between the signal intensities of DNA from undigested and digested minichromosomes of hypoxic cell cultures was found (Fig 4) This indicates that significant unwinding does not occur under hypoxia Smaller local distortions, which were detected after binding of T antigen to SV40 site II at the early palindrome and the AT-rich region in vitro [6–11], may exist under hypoxia They may, however, be protected against digestion by micrococcus nuclease through the origin-bound T antigen hexamer In vitro DNase protection assays demonstrated that the entire 64-bp core origin is protected against digestion by DNase I, when T antigen is bound [6,35] After re-oxygenation, the minichromosomes became more sensitive to nuclease P1 digestion, resulting in an increase of nicked open circle DNA and in a decrease of supercoiled DNA compared to the undigested fraction P1 susceptibility of the minichromosomes became apparent just after re-oxygenation and remained constant for longer re-oxygenation times, indicating that the length of the unwound DNA stretch is not decisive for P1 digestion in Ó FEBS 2002 Initiation of SV40 replication in vivo (Eur J Biochem 269) 2389 Fig Dissociation of minichromosome-bound RPA-34 by excess competitor single-stranded DNA Minichromosomes of hypoxic or re-oxygenated SV40-infected cell cultures were incubated without (A) or with (B) 40 lgỈmL)1 of denatured herring sperm DNA for 30 at 30 °C Minichromosome-bound proteins were isolated, separated by SDS/PAGE and Western blotted The RPA subunits were detected with specific antibodies Exposure times were the same for (A) and (B) Re-oxygenation times are indicated H, hypoxic cells Fig Sensitivity of minichromosomes of hypoxic and re-oxygenated cell cultures to single-stranded DNA-specific nuclease P1 Aliquots of SV40 minichromosomes of hypoxic or re-oxygenated cells were immunoprecipitated with immobilized T antigen-specific antibody with or without prior digestion with P1 nuclease (1 U) SV40 DNA was isolated, separated on an agarose gel, blotted and detected by a 32 P-labeled DNA-probe Signal intensities of open circle and supercoiled SV40 DNA-bands were quantified by densitometric evaluation The signal intensities of the undigested aliquots were set as 100% d, signal intensity of open circle DNA (oc); j, signal intensity of supercoiled DNA (sc) H, hypoxic this test Only about 20–30% of the minichromosomes were sensitive to nuclease P1 digestion As digestion of re-oxygenated minichromosomes with one tenth of nuclease P1 activity yielded the same amount of open circle DNA (data not shown), it may be concluded that the digestion was complete This again indicates that only a minor fraction of the immunoprecipitable minichromosomes was actually engaged in replication after re-oxygenation RPA-34 is dissociated from hypoxic minichromosomes by addition of single-stranded competitor DNA RPA-34 was found to be associated with hypoxic minichromosomes, despite of the facts that RPA-70 was barely detectable (Fig 2) and that the viral genome was obviously not unwound before re-oxygenation (Fig 4) As the interaction between RPA and single-stranded DNA is largely mediated by RPA-70 [36,37], these observations together indicate that RPA-34 is bound to the viral chromosome by protein–protein interactions or by any other means than interaction with single-stranded DNA under hypoxic culture conditions Following re-oxygenation, part or all of the prebound RPA-34 may become integrated into the RPA heterotrimer complex In order to examine which amount of RPA-34 was bound to unwound SV40 minichromosome DNA in form of a RPA heterotrimer complex before and after re-oxygenation, we made use of the fact that, once bound to single-stranded DNA, the resulting complex cannot be disrupted by excess single-stranded competitor DNA [28] RPA-34 bound by other means to the SV40 minichromosome, on the other hand, is readily displaced by excess competitor DNA (see below), probably because it contains a single-stranded DNA-binding motif [38,39] We therefore treated minichromosomes isolated from hypoxic or re-oxygenated cell cultures with an excess of single-stranded herring sperm DNA (Fig 5B) and compared the dissociation of RPA-34 with the respective untreated control (Fig 5A) RPA-34 was found to be almost totally displaced from minichromosomes of hypoxic cell cultures Minichromosomes from cells re-oxygenated for longer than min, on the other hand, retained part of their RPA-34 Higher phosphorylated forms of RPA-34 were almost undetectable, as shown in Fig 5, indicating that they were probably not resistant to incubation at 30 °C under the chosen conditions Note that isolation of minichromosomes was usually performed at °C throughout Alternatively, a phosphatase may have dephosphorylated RPA-34 during the incubation Minichromosome-bound RPA-70 was resistant to displacement by single-stranded competitor DNA (data not shown) Altogether, the results indicate that from only after re-oxygenation onwards, i.e the time we have detected significant unwinding of the SV40 genome [5], an increasing amount of RPA-34 is bound to the viral minichromosome in the form of a single-stranded DNAassociated RPA trimer complex Phosphorylation of the 34-kDa subunit of RPA is not essential for unwinding of the viral minichromosome As mentioned above, the 34-kDa subunit of RPA is additionally phosphorylated immediately after re-oxygenation Phosphorylation of p34 has been suggested to influence initiation efficiency of SV40 DNA replication [40] under hypoxia In vitro, however, SV40 replication proceeds normally, irrespective whether RPA-34 is phosphorylated or not [38] Among the kinases described so far to phosphorylate RPA-34 in vivo are cdk2-cyclin A, cdc2cyclin B, ataxia telangiectasia-mutated (ATM), and DNAdependent protein kinase [32,33,41–43] These kinases are effectively inhibited by staurosporine [44,45] or olomoucine [45,46], and wortmannin [47], respectively To examine whether phosphorylations catalysed by the above mentioned kinases are essential for initiation of SV40 replication after re-oxygenation, we tested the influence of these inhibitors on the formation of SV40 form U, which Ó FEBS 2002 2390 H.-J Riedinger et al (Eur J Biochem 269) was shown to be a product of the unwinding of the viral origin region in vivo [5] The inhibitors were added to the hypoxically cultivated cell cultures 30 before re-oxygenation After re-oxygenation for min, whole-cell DNA was isolated and separated on a chloroquine containing gel Form U was detected after blotting by a SV40-specific probe Figure 6A shows that none of the inhibitors suppressed formation of form U, indicating that, after re-oxygenation, phosphorylations catalysed by protein kinases which are sensitive to staurosporine, olomoucine or wortmannin are not essential for unwinding In a further experiment, we examined the effect of the same inhibitors on phosphorylation of RPA-34 after re-oxygenation (Fig 6B) Staurosporine and olomoucine had no influence on the phosphorylation of RPA-34, whereas wortmannin inhibited formation of all higher phosphorylated p34-species As only ATM and DNAdependent protein kinase are described so far to phosphorylate RPA-34 and are sensitive to wortmannin, it seems likely that one or both of them phosphorylate(s) RPA-34 after re-oxygenation However, other wortmannin-sensitive protein kinases cannot be excluded DISCUSSION Fig Effect of different protein kinase inhibitors on generation of SV40 form U and phosphorylation of RPA-34 after re-oxygenation Staurosporine (10 lM), olomoucine (100 lM) or wortmannin (20 lM) were added to SV40-infected CV1 cells 30 before re-oxygenation After re-oxygenation for min, SV40 DNA (A) or minichromosomebound proteins (B) were isolated SV40 DNA was separated on a chloroquine containing agarose gel, blotted and detected by hybridization against a SV40-specific DNA probe Minichromosome-bound proteins were separated by SDS-PAA gel electrophoresis and Western blotted The p34 subunit of RPA was detected with a specific antibody 1, hypoxic cells, not re-oxygenated; 2, cells re-oxygenated without inhibitor; 3, cells re-oxygenated in the presence of staurosporine; 4, cells re-oxygenated in the presence of olomoucine; 5, cells re-oxygenated in the presence of wortmannin LC, late Cairns SV40 DNA; IC, intermediate Cairns SV40 DNA; T, topoisomers of mature SV40 DNA (form I); U, form U Initiation of SV40 DNA replication is inhibited in cells cultivated under hypoxic conditions The hypoxic block was shown to be situated before the unwinding of the viral origin region and primer synthesis by polymerase a-primase [4,5] After re-oxygenation, unwinding and primer synthesis were detectable within 3–5 followed by an almost synchronous round of viral replication The present study intended to further characterize the hypoxia-induced inhibition of unwinding by analysis of the binding of distinct replication proteins to the SV40 minichromosome before and after re-oxygenation We found that T antigen, RPA-34, topoisomerase I, primase-48, RFC-37, and polymerase d-125 were bound to the viral minichromosome already before re-oxygenation RPA-70, polymerase a-180, and PCNA were barely detectable under hypoxia and increased significantly after re-oxygenation (Fig 2) The small amounts of these proteins, which were detectable under hypoxia, may be bound to originally replicative viral chromosomes, which were no more replicated during the hypoxic period [4] These chromosomes amount up to 5% of the replicationcompetent viral chromatin In vitro studies on SV40 DNA replication suggest that the following steps occur during initiation First, in an ATPdependent reaction, SV40 large T antigen binds as a double hexamer to the viral origin, leading to local distortions in the AT-rich region and partial melting of the early palindrome [7–11] The denatured DNA in the early palindrome is then bound by RPA, possibly at the pyrimidine-rich top strand [8,13,48,49] RPA is first associated in form of an unstable complex (RPA8nt), in which it binds eight nucleotides of single-stranded DNA [13,30,31] After further unwinding of the origin, including part of the central palindrome, RPA8nt turns into a stable complex (RPA30nt), in which RPA contacts about 30 nucleotides of single-stranded DNA Formation of the RPA30nt-complex may be followed by phosphorylation of the RPA-34 subunit [30,31,49] Partial denaturation of the central palindrome leads to loss of interaction of T antigen with its recognition sequence at site II and probably to activation of its helicase activity [49] This in turn may initiate more extensive unwinding of the viral genome, followed by primer synthesis and elongation Supposing that in vivo initiation of SV40 replication proceeds similarly, association of T antigen with the SV40 genome (Figs and 3) indicates that the first step of initiation, i.e recognition and binding of the viral origin, is not inhibited under hypoxia Figure indicates that after re-oxygenation, a significant part of the minichromosome-bound T antigen remains associated with the core origin In accordance with this Ó FEBS 2002 Initiation of SV40 replication in vivo (Eur J Biochem 269) 2391 result, KMnO4 footprinting experiments of the SV40 core origin region revealed no differences between hypoxic and re-oxygenated minichromosomes (data not shown) What may be the reason that T antigen remains bound to the SV40 origin after initiation of replication? Several explanations are possible First, initiation of replication upon re-oxygenation is not exactly synchronous Secondly, only a part of the T antigen-containing minichromosomes actually replicate, and thirdly, the duplicated origin may be rebound by free T antigen, leading to restoration of the state before re-oxygenation The last mentioned notion is supported by the observation that, after a significant decrease after re-oxygenation, minichromosome-bound T antigen was found to be re-elevated and after re-oxygenation (Fig 2) Unwinding of the viral origin, primer synthesis and primer elongation are dependent on re-oxygenation This has been shown previously [5] and is also demonstrated by the results presented here The data indicate that unwinding is initiated as soon as after re-oxygenation (a) The minichromosomes were sensitive to single-stranded DNA-specific nuclease P1 at this time (Fig 4) (b) RPA-70, which is responsible for binding of RPA to single-stranded DNA [36,37], increased slightly as soon as after re-oxygenation (Fig 2) (c) Additional phosphorylations of RPA-34, probably catalysed by ATM and/or DNAdependent protein kinase, were detectable after re-oxygenation (Fig 2) The fact that at least some of these phosphorylations depend on RPA’s binding to singlestranded DNA [30–34] points to the beginning of unwinding Extensive and constant primer RNA-DNA synthesis does not seem to occur before after re-oxygenation This is suggested by the fact that minichromosome-bound polymerase a-180 increased significantly just after reoxygenation and then remained unchanged (Fig 2) Primer synthesis may depend on larger single-stranded bubbles which are only attained after Consistently, RPA-70 increased until and the fraction of RPA-34, which could not be displaced by addition of single-stranded competitor DNA, i.e the fraction which was probably complexed in single-stranded DNA bound RPA [28], increased until after re-oxygenation (Fig 5) PCNA binding and stabilization of polymerase d-125 against degradation, probably indicating primer elongation, were detectable after re-oxygenation and further increased until (Fig 2) Considering the binding behaviour of all proteins examined, it is noticeable that some of them are stably bound to the minichromosome only at the moment they are actually needed (RPA-70, polymerase a-180, PCNA), whereas others are present in minichromosomes already under hypoxia (RPA-34, topoisomerase, primase-48, RFC-37, polymerase d-125) Remarkably, individual subunits of protein complexes, generally believed to act only in a complexed form, e.g RPA-34 and -70, primase and polymerase a, or RFC, PCNA and polymerase d, behave differently in this respect Murti et al [50] found that the RPA subunits also partition differently during the cell cycle The authors demonstrated that the subunits colocalized only during the G1- and S-phase of the cell cycle During mitosis, the subunits dissociated and partitioned into different cell compartments; p34 was found at the chromosomes, p70 at the spindle poles and p11 in the cytoplasm Despite of the fact that hypoxic SV40-infected cells probably never leave a S-phase-like state, these results show that the different subunits of RPA are not necessarily assembled in the heterotrimer complex but may also exist as single proteins in the living cell Altogether, the presented results situate the hypoxiainduced block of SV40 replication somewhere between binding of the T antigen to site II and unwinding of the viral origin The mechanism, which triggers the release of the block following re-oxygenation, remains unclear Clearly, the period between re-oxygenation and beginning of unwinding is too short to allow changes in gene expression or other time-consuming processes The possibility that some proteins have to be synthesized before initiation is ruled out by the fact that inhibition of protein biosynthesis by emetine immediately before re-oxygenation has no influence on unwinding and the following viral replication round (data not shown) Moreover, all replication proteins that were not associated with the viral minichromosome under hypoxia (i.e RPA-70, polymerase a-180, PCNA) were detectable in the cell lysate of hypoxic SV40-infected cells by the respective antibodies (data not shown) This indicates that the proteins necessary to replicate the SV40 genome are present under hypoxia One mechanism to prevent premature initiation may be sequestration of essential replication proteins The protein subunits, which are not detectable in hypoxic minichromosomes, may be bound to other proteins and thus excluded from association with the SV40 chromatin as long as hypoxia lasts Alternatively, the minichromosome-bound subunits may interact with other (minichromosome-bound) proteins through a domain necessary for complex formation under hypoxia In principle, both possibilities can prevent formation of functional protein complexes, like the RPA heterotrimer or polymerase a-primase After re-oxygenation, these unproductive protein–protein interactions may be resolved, by so far unknown mechanisms, in favour of functional proteins, which then initiate viral DNA synthesis The active inhibition of complex formation may be part of a mechanism that prevents uncontrolled unwinding and replication under hypoxia or otherwise unfavourable conditions Besides this, other possibilities are conceivable Unwinding may depend on dephosphorylation of serine residues 120 and 123 of T antigen by protein phosphatase 2A Dephosphorylation was shown to be necessary for T antigen-catalyzed origin unwinding in vitro [51,52] Binding of T antigen to the viral origin as a double hexamer, on the other hand, was possible irrespective of whether Ser120 and 123 were phosphorylated or not Effective unwinding is also influenced by phosphorylation of Thr124 of T antigen, probably catalysed by cyclin A/cdk2 [53–56] As neither staurosporine nor olomoucine, both inhibitors of cyclin A/ cdk2, inhibit formation of form U, i.e unwinding, after re-oxygenation (Fig 6A), it seems likely that Thr124 is phosphorylated before re-oxygenation Alternatively, binding of transcription factors like AP1 or NFjB may be necessary to activate unwinding after re-oxygenation [57–61] In a recent study, we have shown that glucose in millimolar concentrations prevents hypoxia-induced inhibition of SV40 DNA replication in infected CV1 cells [62] As we have outlined in this study, though O2 and glucose are main substrates for cellular ATP generation, it seems 2392 H.-J Riedinger et al (Eur J Biochem 269) unlikely that ATP shortage is responsible for inhibition of SV40 replication under hypoxia This was suggested by the finding that the ATP concentration remained unchanged in SV40-infected CV1 cells during a 4-h hypoxic incubation, whereas unwinding, as determined by generation of SV40 form U, and incorporation of [methyl-3H]deoxythymidine into DNA declined to below 20% of control 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Baeuerle, P.A (1995) The genomic response of tumor cells to hypoxia and reoxygenation Differential activation of transcription factors AP-1 and NF-jB Eur J Biochem 234, 632–640 62 Riedinger, H.J., van Betteraey-Nikoleit, M., Hilfrich, U., Eisele, K.H & Probst, H (2001) Oxygen-dependent regulation of in vivo replication of simian virus 40 DNA is modulated by glucose J Biol Chem 276, 47122–47130  ... after re-oxygenation Phosphorylation of p34 has been suggested to influence initiation efficiency of SV40 DNA replication [40] under hypoxia In vitro, however, SV40 replication proceeds normally,... (B) Re-oxygenation times are indicated H, hypoxic cells Fig Sensitivity of minichromosomes of hypoxic and re-oxygenated cell cultures to single-stranded DNA-specific nuclease P1 Aliquots of SV40. .. DNA labelled by addition of [2-14C]deoxythymidine (5 nCiỈmL)1) immediately after infection of CV1 cells with SV40 covalently closed, supercoiled SV40 DNA was visible Accordingly, this peak was