JOURNAL OF VIROLOGY, Jan. 1990, p. 347-353 Vol. 64, No. 1 0022-538X/90/010347-07$02.00/0 Copyright C 1990, American Society for Microbiology Programmed Factor Binding to Simian Virus 40 GC-Box Replication and Control Sequences ROBERT L. BUCHANANt AND JAY D. GRALLA* Department of Chemistry and Biochemistry and Institute, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, California 90024 Received 6 July 1989/Accepted 22 September 1989 Nuclear footprinting revealed a temporal program involving factor binding to the repetitive GC-box DNA elements present in the simian virus 40 regulatory region. This program specified ordered and directional binding to these tandem regulatory sequences in vivo during the late phase of infection. The program was interrupted by the DNA replication inhibitor aphidicolin or by inactivation of the viral replication factor simian virus 40 T antigen, suggesting a link between viral DNA replication and new factor binding. Measurements of DNA accumulation in viruses lacking either the distal or proximal halves of the GC-box region suggested that the region has a dual role in replication control. Overall, the data point to important relationships between DNA replication and factor binding to the GC-box DNA, a multifunctional regulatory region.

Factor binding to mammalian regulatory DNA elements is MATERIALS AND METHODS of central importance in gene control. The cellular distribu- Viral infections, preparation of nuclei, and DNA purifica- tion and activity of such factors likely contributes to cell- tion. SV40 strain tsA58 and parental strain VA45-54 were specific patterns of gene expression. Although many DNA- obtained from Peter Tegtmeyer. In general, CV-1 cells were binding factors have been identified (see reference 26 for an infected with 1 to 5 PFU of SV40 per cell and maintained in example), the mechanisms that control their distribution and Dulbecco modified Eagle medium plus 2% calf serum plus activity are not understood. Many models relevant to such antibiotics. Enough cells were infected to provide approxi- mechanisms center on programmed factor binding to DNA mately 0.1 to 0.5 ,ug of SV40 DNA per footprinting reaction. and especially on the influence of DNA replication on such Isotonic nuclei lysed with Nonidet P-40 (Sigma Chemical programs (see, for example, reference 5). Co.) were prepared and nicked with DNase I as previously Study of mammalian DNA tumor viruses has revealed described (6). DNA was purified as previously described (6), well-defined genetic programs that depend directly or indi- except that the order of RNase A and proteinase K diges- rectly on DNA replication (45). In simian virus 40 (SV40) tions was reversed to use endogenous RNA as a carrier in and adenovirus, components of the replication and transcrip- DNA precipitations. tion machinery involved in such programs have been purified In some experiments, DNA replication was inhibited (14, 26). Most of the protein components are host cell before isolation of infected nuclei. At 32 h postinfection, the proteins, which suggests that regulation of viral programs cell culture medium was brought to 5 ,ug of aphidicolin has important features in common with cellular regulation. (Sigma) per ml and infections were continued for up to 16 h Among these proteins are cell factors which control SV40 (29). replication and transcription through common DNA ele- Primer extension and gel electrophoresis. The sites of ments (10). A critical DNA target of these cellular proteins is DNase I nicking were determined by hybridizing 5'-end32P- the repeated motif (GGGCGG)6 or the GC box which is a labeled oligonucleotide primers to denatured samples and regulatory element adjacent to the viral core origin of extending them to breaks with Klenow DNA polymerase replication that influences DNA replication (24, 31, 32), as essentially as previously described (6, 20), except that all well as early (1, 11) and late transcription (4, 13). The role of extension reactions were done at 50 to 52°C. Base denatur- these factors in uninfected cells is also likely to be involved ation of viral DNA templates gave a lower background than with gene regulation, since GC boxes are often associated heat denaturation for samples isolated before 40 h. Base with regulatory DNA (27, 38). denaturation and subsequent primer extension have already In situ DNase I footprinting of nuclear SV40 templates been reported (3). isolated from infected CV-1 monkey cells showed abundant Quantitation of replicated viral DNA. Confluent CV-1 cells factor binding to the SV40 GC boxes at 48 h postinfection, were infected with equal titers of wild-type SV40 mutant but neither the identity ofthe factors nor their functional role virus strain 776, SV-P7, or PXS7. After 2 h, infected plates is known (6). In this report, we identify a genetic program were washed three or four times with warm Dulbecco involving this factor and present experiments suggesting that modified Eagle medium to reduce the amount of unattached the programmed binding requires templates competent to virus. Viral DNA was prepared as described above and replicate. Since GC boxes and repetitive DNA are relatively electrophoresed on 1% agarose gels after linearization with common in cells, these observations may have interesting EcoRI. Full-length nick-translated ([32P]dATP) FIII SV40 implications for programming interactions with cellular reg- DNA was used to probe DNA from 12- to 24-h postinfection ulatory DNA. samples by Southern hybridization. DNA that hybridized to the labeled SV40 probe was visualized by autoradiography * Corresponding author. with Cronex film at room temperature. Several autoradio- t Present address: Division of Biology 156-29, California Institute grams at different exposures were scanned by densitometry. of Technology, Pasadena, CA 91125. Viral DNA samples prepared from experiments done at 24 347 348 BUCHANAN AND GRALLA J. VIROL.

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11 III IV v v I AATT AGTCAGCCAT GGGGCGGAGA ATGGGCGGAA CTGGGCGGAG TTAGGGGCGG GATGGGCGGA GTTAGGGGCG GGACTATGGTT 28 31 41 51 61 71 81 91 101 FIG. 1. The SV40 control region with oligonucleotide primers used in footprinting experiments. The bidirectional arrow designates the linear SV40 DNA sequence with nucleotide numbers according to Tooze (45). The directions of early and late transcription are indicated. Relevant SV40 control regions are also shown, including the centers of three T-antigen (T AG)-binding domains. Three arrows labeled 21 depict the six GC-box regulatory elements. The wedge labeled 72 is the origin-proximal portion of the 72-base-pair viral element. Oligonucleotide primers are shown as arrows with numbers (5145 and 5230) indicating the 3' ends of the primers. At the bottom, the SV40 GC-box region is expanded to show the nucleotide sequence from nts 28 to 111. Within this expanded region, the GC boxes are numbered with roman numerals. ORI, Origin of replication. to 65 h postinfection were linearized and electrophoresed on was partially protected from DNase I digestion by a bound 1% agarose gels after dilutions typically in the range of 1:10 factor at 40 h postinfection (6). The technique detects only to 1:1,000. DNA was visualized by ethidium bromide stain- the interaction of abundant factors with nonencapsulated ing and photographed with Kodak Tri-X film. The amount of DNA; the approximately 90% of SV40 DNA in virions and DNA in each lane was determined by densitometry. A range previrions is relatively DNase I resistant (see reference 6) of exposures were taken to assure linear response. and does not contribute significantly to the signal in this assay. Factor binding to less than half of the remaining 10% RESULTS of the DNA is difficult to detect with this method (6). The late phase of the SV40 life cycle involves programmed Probing the replication core region in vivo. Figure 2 shows production of viral DNA and protein with the goal of the results of in situ probing of the replication core se- assembling infectious virus (45). As the late phase proceeds, quences and the adjacent T-antigen-binding site I. Factor is assessed of DNA com- more RNA is produced from the expanding pool of DNA, binding by comparing digestion with 2 to but the average transcriptional activity of SV40 DNA tem- plexed protein (nuclei, lanes 4) with digestion of plates remains constant (18). However, the average replica- naked DNA (in vitro, lane 1). There was significant protec- tion activity of SV40 DNA decreases during this time (24, tion of sequences over T-antigen site I at 32, 40, and 48 h 33). Thus, instead ofthe exponential production of DNA that postinfection. However, strong protection of sequences cor- would accompany uncontrolled replication, the amount of responding to the core elements for replication which are SV40 DNA simply increases linearly. This relationship be- adjacent to this site was not observed (compare lanes 2 to 4 tween replication and transcription apparently ensures a with lane 1). balanced ratio of protein to DNA. The purpose of these These footprinting results are in excellent accord with initial experiments was to probe the in situ interactions with long-standing observations in the literature. At this time in DNA elements near and within the replication core during the lytic cycle, the dominant interaction in this region is the time when the programmed decrease in average template expected to involve T antigen bound at site I acting as a activity occurs. of early transcription (34). Immunological studies The method of probing DNA-protein interactions (6, 20) indicated that approximately 10% of SV40 DNA in vivo is involves adding DNase I to nuclei isolated from SV40- bound by T antigen (42), and this agrees well with strong infected CV-1 cells to lightly nick the endogenous SV40 protection of approximately 10% of the templates in these DNA. The extrachromosomal SV40 DNA is then isolated nuclear footprints. The limits of protection seen in these and denatured to produce strands whose ends were formed nuclear experiments are similar to limits of T-antigen binding by DNase I nicking in situ. The attack pattern is revealed by to site I in vitro (34, 44). Replicating templates constitute hybridizing 5'-end 32P-labeled DNA primers adjacent to the only 1% of SV40 DNA (42), and thus, protection of the region of interest and extending them in vitro to reveal stop replication core origin in this proportion of templates would sites corresponding to nicks (20). Primer 5145 is designed to be well below the level of detection; no extensive protection probe the core replication elements, and primer 5230 reads was seen, even with lighter exposures of the autoradiogram. through the accessory GC-box element (Fig. 1). Previously, The time course of probing revealed no changes that could probing with primer 5230 showed that the GC-box region easily account for the programmed decrease in template VOL. 64, 1990 PROGRAMMED GC-BOX BINDING AND DNA REPLICATION 349

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OmM.F Om FIG. 3. Nuclear footprints produced on the late mRNA template or C strand in the GC-box region. Nuclei from SV40-infected cells were prepared at various times postinfection and nicked with approximately 0.01 ,ug of DNase I per ,ul for 2 min (lanes 2, 3, and 5 to 7). Nuclear DNAs were purified, denatured, and used as templates for hybridization with oligonucleotide primer 5230. Lane C depicts control SV40 DNA nicked in vitro with approximately 0.0001 p.g of 1 2 3 4 DNase I per ,ul (lanes 1 and 4). The numbers above lanes 2, 3, and 5 to 7 indicate the times (hours) postinfection at which nuclei were FIG. 2. Nuclear protection of the region near the replication probed. SV40 nucleotide numbers are shown to the left of each gel, origin (ORI) was probed on the late mRNA template strand with along with the locations of the GC-box 21-base-pair repeat elements primer 5145 at 32, 40, and 48 h postinfection. The minimum (left panel) and individual GC boxes (right panel). replication origin, which is approximately coincident with T-antigen (T Ag)-binding site II, is indicated, as is site I. Lane 1 contained SV40 nicked in vitro and probed with primer 5145 as a control (C), as strong; this weakening occurred principally between nts while lanes 2, 3, and 4 contained samples nicked in nuclei at 32, 40, 70 and 100. Even earlier, at 32 h postinfection (lane 5), this or 48 h postinfection. region between nts 70 and 100 was much less well protected, and at 24 h postinfection, the weak protection of this region was diminished further (lane 2). This loss of protection was replication activity during late times. The only change seen accompanied by the appearance of two positions of DNase I was that the internal A+T-rich spacer region within T-antigen hyperreactivity near nts 75 and 95. As discussed above, site I was slightly more protected as the infection proceeded. strongly protected regions in this assay are taken to reflect Programmed binding to multiple GC boxes in vivo. A quite factor binding to about 10% of the nuclear SV40 molecules. different result was seen when the accessory GC-box DNA This is an order of magnitude higher than the fraction of element was probed (Fig. 3). When DNA samples from 48 h templates actively engaged in either replication or transcrip- postinfection were probed, strong protection was seen over tion (15, 42). the entire GC-box regulatory element. When this pattern Thus, our view of this program can begin at 24 h postin- (lane 7) was compared with that of the in vitro control (lane fection, when replication activity is still high (24, 33). At that 4), it was found that all bands between approximately time, only the region surrounding GC boxes 2 and 3 is nucleotides (nts) 50 and 100 were strongly protected from strongly protected. As the temporal program proceeds, GC DNase I digestion in SV40-infected nuclei. The region be- boxes 4 to 6 are bound progressively with the factor. This tween nts 30 and 50 was partially protected, and only weak ordered binding has not been reported for any of the cellular protection was evident within the enhancer (see also refer- factors known to bind the GC-box region. As reported ence 6). At the earlier time of 40 h postinfection (lane 6), the previously (see Fig. 3 in reference 6), this factor protects the pattern was similar, except that the protection was not quite early mRNA or G strand less strongly in the GC-box region, 350 BUCHANAN AND GRALLA J. VIROL. another property not noted with isolated GC-box DNA transcription factors. This preference for the late mRNA or C strand was maintained throughout the late phase (data not programmed factor binding ex- .n 3i shown). We conclude that 4-) the becomes less 32 C 4' O 4 O 4; a t-, ists; during the time when pool of templates 'IT active in producing DNA, the GC-box DNA is increasingly :1 bound by an abundant nuclear factor. Inhibition of programmed factor binding inhibition of DNA replication. We began experiments to probe the mechanism . underlying this progressive factor binding to the GC-box DNA region. A key issue involving genetic programs in SV40 and in cells is whether DNA replication plays a direct

yv. a>t role in influencing factor binding (5, 45, 47, 48). This issue :e :# can be explored in this system because there is continuous *.. #. .. production of new templates while the SV40 program pro- ,s, ...,. ceeds. Thus, replication can be inhibited early in the pro- vA&g gram and it can be observed whether the programmed :g '.; increase in binding is also inhibited or whether the pattern is F otherwise perturbed. 139 11.. t The possibility that DNA replication is required for factor (5...cr~~~~~~~~cz , .. 3sF rc 4:A. ., M binding was tested initially by inhibiting DNA synthesis with .. aphidicolin (29) and monitoring alterations in factor binding z <'h by nuclear footprinting. In this experiment, a series of plates was infected with SV40. At 32 h postinfection, half of the plates were treated with 5 ,g of aphidicolin per ml. Both

:-. XS control plates and drug-treated plates were incubated for an r.s;. additional 8 or 16 h. At these times (40 and 48 h postinfec- tion, respectively), nuclei were prepared and the SV40 DNA to estimate factor binding. As a was footprinted in situ .t. further comparison, control samples from 32 h postinfection were processed in parallel. Thus, the effect of the DNA synthesis inhibitor aphidicolin on factor binding can be deduced from this collection of data (Fig. 4A). *<}s The results showed that inhibition of DNA replication with aphidicolin inhibited binding of factors to additional sites within the GC-box region. When DNA synthesis was inhibited at 32 h postinfection, continued incubation until 40 or 48 h postinfection did not lead to significant additional factor binding; that is, the 32-h pattern was nearly frozen (Fig. 4A, compare lanes 1, 4, and 5). In parallel samples in FIG. 4. Inhibition of GC-box factor binding by inhibition of DNA which DNA synthesis was not inhibited, the expected in- replication. (A) Nuclei were harvested at 32, 40, and 48 h postin- crease in factor binding occurred during this time (lanes 2 fection, as indicated above the lanes. A plus or minus indicates and 3). These results also indicate that previously bound whether aphidicolin (5 j±g/ml) was added to infected plates at 32 h GC-box interactions were stable and that nonreplicating postinfection. At the indicated times, nuclei were prepared, nicked 16 h in vivo with DNase I, and probed with primer 5230. (B) Cells infected with DNA remained bound by factors for at least VA45-54 (lane 1) or tsA58 (lane 2) for 40 h at 32°C. At 40 h, infected (compare lanes 1 and 5). Similar results were obtained with cells were shifted to 41.5°C (nonpermissive for tsA58 viral replica- cycloheximide, which is known to inhibit protein synthesis tion) and incubated for an additional 10 h at 41.5°C. Nuclei were the and SV40 DNA replication (data not shown). prepared, nicked with DNase I, and probed with primer 5230. The Since aphidicolin inhibits both SV40 (9) and cellular DNA amount of viral DNA present at 40 h corresponded approximately to synthesis, the effect of template replication on factor binding the amount that appeared in a 37°C wild-type infection at 24 h was assessed by using a viral mutant in which cellular postinfection. processes are not affected. In this experiment, parallel cultures were infected with either tsA58 virus or VA45-54, the wild-type parent of mutant tsA58 (43). tsA58 is a virus was blocked (tsA58 in lane 2B). That is, the GC boxes were with a temperature-sensitive T antigen, and thus, it cannot much more protected (lane 1B) when T-antigen-dependent replicate at 42°C. The two viruses were grown at 32°C for 40 viral replication was allowed to continue. The experiment h to allow replication and partial factor binding to occur, demonstrated that SV40 T antigen is required for GC-box both of which occur rather slowly at 32°C. The VA45-54- and filling. Taken together with the inhibition of binding by tsA58-infected cultures were then shifted to the nonpermis- aphidicolin, the data indicate that T-antigen-dependent viral sive temperature of 41.5°C and incubated for 10 h longer. DNA replication is required for programmed factor binding. Viral DNA replication during the 10 h occurred only in Effects of the region containing GC boxes on replication VA45-54-infected cultures. After this incubation, nuclei timing and rate. Previous studies have shown that deletions were prepared from both cultures and footprinted to assess within the GC-box region lead to lowered levels of replicated factor binding (Fig. 4B). This comparison showed that SV40 DNA (23, 24, 32, 50). Since the data indicate that additional factor binding at 41.5°C occurred when viral binding to the GC boxes depends on functional T antigen, it replication proceeded (VA45-54 in lane 1B) and not when it seemed possible that the GC boxes could also influence how VOL. 64, 1990 PROGRAMMED GC-BOX BINDING AND DNA REPLICATION 351

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T antigen turns on replication. The footprinting procedures z are not sensitive enough to probe the onset of replication at 6 early times directly. Instead, we used mutants to investigate SV-P7 the role of the GC-box region in replication timing. Trans- since the 0 fected plasmids are unsuitable for this purpose, E time at which they begin replication is altered (8). Thus, 4c mutants built into viable virus must be used. We had 4 available two such mutant viruses, each of which deletes a different half of the GC-box region. Virus SV-P7 lacks GC boxes 1 to 3 and nine adjacent nucleotides (nt 32 to 73), approaching but not impinging upon the replication core (Buchanan and Gralla, unpublished data), and virus PXS7 2 (16) lacks GC boxes 4 to 6 (nt 73 to 100). Taken together with PXS7 wild-type SV40, the set of viruses allows assessment of the function ofthe 40-base-pair region adjacent to the replication core and the 30-base-pair region between this region and the enhancer. 0 In the first series of experiments, the time of the onset of 10 20 30 40 50 60 70 80 replication was measured in viruses lacking either early-side (SV-P7) or late-side (PXS7) GC boxes. DNA was harvested every 2 h from 12 to 24 h postinfection, and its amount was estimated by probing Southern blots (Fig. 5). The two mutant viruses began to replicate at grossly different times. Time Post-infection (hours) SV-P7 DNA was visible at 12 h postinfection (lane 2) and FIG. 6. Accumulation of wild-type (wt) SV40, SV-P7, and PXS7 gradually increased in amount over the time assayed (lanes from 24 to 65 h postinfection. DNAs from infected plates were 2, 5, 8, etc.). By contrast, PXS7 DNA (lanes 3, 6, 9, etc.) was harvested and quantified at the indicated times postinfection. not visible until 22 h postinfection (lane 18), when it began to accumulate. Thus, deletion of the two halves of this region had very different effects on the timing of DNA accumula- More generally, the 70-base-pair region covered by these tion. This differential effect on replication timing is unlikely two deletions acts as a timing element influencing the onset to be due to altered early accumulation of T antigen, since of SV40 DNA replication. deletion of the late-side GC boxes should not be more The amounts of DNA made by these three viruses were deleterious to T-antigen expression than is deletion of early- also monitored past 24 h postinfection to observe the long- side GC boxes. term effect on rate of DNA production. All three viruses These same blots show that wild-type DNA (lanes 1, 4, 7, accumulated DNA from 24 to 65 h postinfection (Fig. 6) but etc.) was barely visible at 12 h postinfection and increased in at different rates. During this time, wild-type virus produced, amount by 16 h postinfection, as expected (45). During this on average, 0.26 p,g of DNA per h, SV-P7 produced 0.18 time interval, the hierarchy in the amounts of DNA was ,ug/h, and PXS7 produced 0.05 ,ug/h. These data agree with maintained as SV-P7 > wild type > PXS7. We conclude that those of various studies showing lowered DNA yields the PXS7 mutation drastically slows the onset of replication, caused by GC-box deletions (16, 24, 32, 50) but are interest- even though the PXS7 early appears to be nearly ing in two respects. (i) SV-P7, which produced more DNA as efficient as wild-type SV40 in vivo (21). The earlier time at than did the wild type before 24 h postinfection (Fig. 5), which SV-P7 DNA began to accumulate was unexpected for produced less by 65 h postinfection (Fig. 6). (ii) Although two reasons. (i) The region deleted is critical for early SV-P7 and PXS7, which delete separate subregions, had transcription (see above references), and thus, one might opposite effects on the onset of replication, they had the have expected a delay due to slower production of T antigen. same qualitative effect on steady-state replication rates; i.e., (ii) As discussed above, other GC-box deletions have led to both reduced the rate of SV40 DNA synthesis. Thus, the lower, not higher, total levels of SV40 DNA, and this lower region contains separate controls for replication timing and ultimate level of DNA was also true for SV-P7 (see below). steady-state replication rate (Fig. 7). Both halves of the Our best quantitative estimate is that PXS7 is delayed for 6 region are required for optimal DNA accumulation, consis- to 8 h and SV-P7 accelerated modestly, by 2 or perhaps 3 h. tent with the idea that when they are occluded by pro- 352 BUCHANAN AND GRALLA J. VIROL.

l I I I I I III IV v vI H A second property of the bound complex may also have 30 interesting implications, since the factor protects one of the DNA strands more strongly than the other (6); upon strand separation during replication, it is possible that this prefer- EARL Y DELAYS ONSET ACCELERATES ONSET ence for one of the strands would be maintained, allowing preferential transmission to one of the two daughter DNA molecules (see also references 41 and 49). Several different factors have been isolated (17, 28, 35, 36) LATE REQUIRED FOR MAXIMUM REPLICATION RATE that bind SV40 GC-box DNA in vitro since the discovery of SP-1 (11). The abundant GC-box factor bound in SV40- FIG. 7. Effect(s) of SV40 GC boxes on viral DNA replication. infected cells is believed to be of cellular rather than viral SV40 GC boxes are schematically shown as six adjacent boxes. origin (7), although a role for late viral proteins cannot be Below are depicted the effects of two GC-box subregions on DNA replication during the early and late phases of the SV40 lytic excluded. At least two previously characterized GC-box- cycle. binding factors share the unusual property of strand prefer- ence. (i) A chicken globin gene factor binds G-string regula- grammed abundant factor binding or when they are deleted, tory sequences with a strand preference similar to that of the replication is inhibited. The two subregions encompassing SV40 GC-box factor (12). (ii) It has recently been demon- the six GC boxes affect the time of replication onset differ- strated that uninfected CV-1 cells contain a factor that ently, implying that before replication onset, an interesting preferentially binds one strand of an SV40 GC box, although but unknown genetic program exists which controls the it does not appear to bind double-stranded DNA (17). It is initiation of DNA replication. not known whether the monkey cell factor detected in this study is analogous to these factors, SP-1, or any of the other DISCUSSION isolated human factors; GC-box-binding proteins have not These experiments have described a temporal program been purified from monkey cells. In both human and monkey involving interactions at the repetitive SV40 GC-box regu- cells, GC-box elements are common (27) and are often latory elements. T-antigen-dependent DNA replication is repeated when present, as in SV40. Thus, cells contain both required for assembly of a complex that protects the entire components of the DNA-factor interaction described here, region from DNase digestion in nuclei. This assembly pro- and therefore, the interesting properties of the SV40 com- ceeds by filling the GC boxes in order, from the origin- plex are likely to be shared by certain cellular genes. proximal toward the origin-distal elements. It is not known These experiments have confirmed that the SV40 GC-box whether the requirement for T-antigen-dependent replication repeat unit influences the SV40 replication rate (see refer- implies that this genetic program requires specific compo- ences in the introduction) and have also shown that subre- nents of the active SV40 replication complex, which is gions control the timing of replication onset (Fig. 7). Since positioned nearby, or simply newly replicated DNA. mammalian cellular origins of replication are poorly charac- The GC-box region consists of repeated sequences, and terized, it is not known whether some origins are associated repetitive elements are known to be associated with tempo- with GC boxes, G strings, or other repeat elements. How- ral control by diverse proteins, such as X repressor and T ever, it is known that the timing of cellular DNA replication antigen (34, 37). In X, the bound sequences have a dual is precisely controlled. The earliest genes to be replicated function and serve as activator sites at low factor concen- are generally the so-called housekeeping genes (25). GC-box trations but mediate repression when occluded at high pro- repeat elements are highly concentrated in this gene type tein concentrations. These data suggest that the interactions (19, 25). Since some proposals link origins of replication with with the SV40 GC-box elements are analogous; i.e., the transcriptional activation (22), it is possible that these cellu- elements serve as activation sites except when occluded by lar GC boxes influence replication as well as gene expression; factors. The amount of SV40 DNA bound by factors at late recall that SV40 GC boxes have this dual effect (10). The data times is much greater than the amount involved in replica- presented here are most easily interpreted as implying that tion or transcription; thus, at these times, the factor appears the onset of viral replication requires removal of a repressor not to activate the templates but rather to occlude them. from early-region-proximal GC boxes I, II, and III (Fig. 7). Recall that SV40 GC boxes are not required to activate This observation could have significant impact on the eval- transcription at late times (24) and that no net activation of uation and extension of models for control of DNA replica- transcription occurs during this time (18). On the basis of this tion onset in eucaryotic cells (2, 22, 30, 39, 40). circumstantial evidence, we suggest that programmed factor binding at late times progressively occludes the GC-box ACKNOWLEDGMENTS replication element to program the gradual decrease in This research was supported by grants from the National Science template replication activity. Foundation and the American Cancer Society. In the GC box and X examples, repeat units are bound in a specified direction as a program proceeds. Since the LITERATURE CITED GC-box units are of very similar DNA sequences but are 1. Barrera-Saldana, H., K. Takahashi, M. Vigneron, A. Wildeman, bound in a specified order, cooperative interactions may be I. Davidson, and P. Chambon. 1985. All six GC motifs of the involved. However, the mechanism underlying directional SV40 early upstream element contribute to promoter activity in GC-box factor binding remains unknown, since none of the vivo and in vitro. EMBO J. 4:3839-3849. 2. Berg, L., factors known to bind this region in vitro were reported as M. Lasky, A. Stenlund, and M. R. Botchan. 1986. having this property of directional binding (26). Repression of bovine papilloma virus replication is mediated by Neverthe- a virally encoded transacting factor. Cell 46:753-762. less, the ability of prebound factors to specify the nearby 3. Borowiec, J. A., L. Zhang, S. Sasse-Dwight, and J. D. Gralla. positioning of new factors only on templates competent to 1987. DNA supercoiling promotes formation of a bent repres- replicate could have an important influence on reprogram- sion loop in lac DNA. J. Mol. Biol. 195:101-111. ming expression in newly made cells (5, 46, 47). 4. Brady, J., M. Radonovich, M. Thoren, G. Das, and N. P. VOL. 64, 1990 PROGRAMMED GC-BOX BINDING AND DNA REPLICATION 353

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