Assembly of the replication initiation complex on SV40 origin DNA
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《核酸研究医学期刊》
Department of Biological Sciences, University of Delaware, Newark, DE 19716-2590, USA
*To whom correspondence should be addressed. Tel: +1 302 831 8547; Fax: +1 302 831 2281; Email: dsimmons@udel.edu
ABSTRACT
The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase /primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.
Introduction
Replication of simian virus 40 (SV40) DNA begins when the virally coded T antigen binds to the SV40 origin and forms a double-hexameric structure . Subsequently, three cellular proteins, replication protein A (RPA) (4–7), DNA polymerase /primase (pol/prim) (8–11) and topoisomerase I (topo I) (12–15), associate with T antigen to generate an initiation complex. After initiation, the majority of DNA synthesis on both leading and lagging strands occurs in response to additional cellular proteins such as polymerase , PCNA and RF-C (16–18). In all, 11 cellular proteins are utilized to fully replicate the virus DNA (19–25). Although all of these proteins have been purified and studied to investigate their role in replication, there is only marginal understanding of the dynamics of the replication process. Surprisingly, there is little information about how the initiation complex itself assembles and how the three cellular proteins work with the T antigen double hexamer to initiate the synthesis of RNA primers and to extend these primers with DNA.
The assembly of the T antigen double hexamer is fairly well understood. A single monomer is believed to bind to pentanucleotide 1 at the origin (see Fig. 1) and associate with others to form a single hexamer (26–29). In addition to pentanucleotide 1, flanking sequences consisting of the early palindrome (EP) region (Fig. 1) are necessary for formation of the first hexamer (27). A second hexamer then forms from individual monomers over pentanucleotide 3 (26,28). These three origin elements (pentanucleotides 1 and 3 and EP) constitute one assembly unit for the construction of a double hexamer (26). A second assembly unit consisting of pentanucleotides 2 and 4 and the AT track can also support formation of a double hexamer, but does so less efficiently. When a double hexamer forms over the origin, 8 bp of the DNA at the EP region are melted and the DNA at the AT track is untwisted (30–35). These two structural changes together are often referred as ‘structural distortion’. Structural distortion is believed to precede the melting of the origin by T antigen’s helicase activity. The mechanism by which this occurs in under intensive investigation in several laboratories and is believed to be accompanied by extensive structural changes in T antigen as well as in the DNA (36).
Figure 1. Sequence of the SV40 origin. The core origin is indicated between the two double triangles and consists of the EP region, site II and the AT track. Arrows within site II (shown in red) represent each of the four GAGGC pentanucleotides that T antigen recognizes. Arrows within the EP region represent an imperfect palindromic sequence. T antigen binding site I (highlighted in green) is shown on the early side of the core origin. Each of its two pentanucleotides is indicated as an arrow. The sequence between the single blue triangles is deleted in pSKORI.
Only recently has it been recognized that topo I is an integral component of the initiation complex (12,15). This enzyme nicks and religates DNA in order to relieve torsional strain ahead of the replication fork, and there is increasing evidence that it functions in combination with T antigen as a gyrase to unwind and relax DNA (12–15,37,38). Very recently, nucleolin has also been implicated as an initiation factor that facilitates assembly and activity of a gyrase complex (38). Although there is no evidence that nucleolin is needed in vitro for DNA replication, it may well be needed in infected cells. Topo I forms a specific interaction with double hexamers and does not bind well to smaller oligomerized forms of T antigen (12). By mapping the sites of interaction between T antigen and topo I, we have recently proposed that topo I binds to the ends of the double-hexamer complex (39). In this orientation, we hypothesize that the nicking and religation activity of topo I is coupled to the unwinding activity of T antigen to efficiently propel the replication fork.
Pol/prim consists of four subunits, of which one (p180) is the polymerase catalytic subunit, one (p68) is a structural unit and the other two (p58 and p48) are components of the primase (40). T antigen binds to the p180 subunit (41) and stoichiometric analysis predicts a ratio of one pol/prim molecule per T antigen hexamer (42). The primase synthesizes 8–10 nt stretches of RNA starting at sites with a consensus sequence of 5'-TTN-3' (43). Pol/prim then extends these RNA primers with 35 nt of DNA (17,44). The enzyme is not very processive and readily falls off its template (40). Further DNA synthesis requires elongation factors DNA polymerase , PCNA and RF-C (6,16,17).
RPA is a three-subunit protein that coats and protects exposed single-stranded regions of DNA at replication forks (45). RPA also has the added function to stimulate pol/prim to lay down RNA primers (46). The largest subunit (p70) has single-stranded DNA binding activity as well as the ability to bind T antigen (47). The binding site for RPA on T antigen has been mapped to residues 164–249 (48). RPA can also bind directly to pol/prim apparently through interactions involving all three RPA subunits (5,49,50).
DNA synthesis can take place in vitro using the so-called ‘monopolymerase system’, which consists of plasmid DNA containing the SV40 origin, T antigen and all three cellular proteins described above (9,17,51,52). Under these conditions, a heterogenous population of DNA molecules is synthesized ranging in size from a few nucleotides to several thousand. Optimal synthesis of DNA is dependent on all four proteins.
In this study, we determined the stoichiometry between T antigen and topo I and evaluated the dynamics of assembly of the initiation complex. We show that DNA polymerase appears to be the first cellular protein to be required for the formation of the initiation complex. However, this enzyme cannot be detected in the complex, although its presence is needed for T antigen double hexamers to remain associated with origin DNA in the presence of topo I and for subsequent proteins such as topo I itself and RPA to bind optimally.
Materials and Methods
Construction of origin DNA fragments
All origin-containing fragments were generated by PCR amplification of the SV40 origin from pSKori (13). Appropriate primers 20 nt long were designed to amplify specific regions of the origin to make the fragments used in this study. These corresponded in sequence to that shown in Figure 1 starting with each arrow. A fragment missing T antigen binding site I was made by PCR from pSKORI, which is identical to pSKori except for a deletion of 31 bp as shown in Figure 1. One PCR primer for this fragment started at –72 and extended beyond the deletion to nucleotide –24. Biotinylated DNA was made by PCR using a ‘down’-biotinylated oligonucleotide whose 5' end was modified with biotin and which corresponds to residue +128 (Fig. 1) and an ‘up’ primer with 5' end at either –64 or –55. DNA fragments were purified by agarose gel electrophoresis.
Purification of T antigen
WT T antigen was purified from baculovirus-infected insect cells by immunoaffinity chromatography as described previously (53) using PAb101 antibody (54). All preparations were >90% pure.
Purification of topo I
WT topo I was purified by standard chromatography from extracts of baculovirus-infected Sf9 cells as described previously (55). Purity was >90%.
Purification of pol/prim
Pol/prim was purified by a novel procedure that permitted the preparation of highly pure and active enzyme in two steps. The procedure was based on the Impact-CN system (New England Biolabs) where one of the subunits was tagged with a modified form of intein. First, the cDNA for the p58 primase subunit of the enzyme was generated by PCR from baculovirus DNA expressing that subunit (the virus was a gift from Dr Ellen Fanning). The PCR primers were designed according to the manufacturer’s directions for insertion into pTYB3 (NEB). The PCR-generated cDNA was digested with XbaI and SapI and ligated into gel-purified linear pTYB3 digested with the same enzymes. Clones were screened for the presence of the insert in the correct orientation by restriction digestion with XbaI and PstI, and potential clones were subjected to DNA sequencing. The DNA of clones with the correct sequence was digested with the same two enzymes and the smaller fragment was ligated into the equivalent sites of p1393 (Pharmingen) baculovirus transfer vector. Recombinant baculovirus was made by cotransfection into Sf9 insect cells as described by the manufacturer (Pharmingen). Expressing virus was screened by immunofluorescence using an antibody directed against the primase subunits (a kind gift from H. P. Nasheuer).
To generate pol/prim, High 5 insect cells were infected with the recombinant virus expressing the intein tagged p58 primase subunit and co-infected with recombinant baculoviruses expressing each of the other three untagged pol/prim subunits (kind gifts from Dr Ellen Fanning and Dr T. S. Wang). At 60 h post-infection, the cells were lysed by dounce homogenization in 0.15 M Tris pH 7.0, 0.15 M NaCl, 0.001 M EDTA, 10% glycerol, 0.5% NP40 and 0.001 M phenyl-methylsulfonyl fluoride (PMSF) and the clarified lysate was incubated with chitin beads. After 1 h at 4°C, the beads were centrifuged and washed twice with 0.05 M Tris pH 7.0, 0.5 M NaCl, 0.001 M EDTA, 10% glycerol and 1% NP40, and twice with the same buffer minus the NP40. The beads were transferred to a small column and two column volumes of cleavage buffer were quickly passed over the column. More cleavage buffer was added and the column was left stoppered in the cold room overnight. The next day, the eluate was recovered into several fractions. Fractions containing protein were pooled and the pol/prim was further purified and concentrated over a small (0.5 ml) column containing hydroxylapatite equilibrated in cleavage buffer lacking DTT. The column was washed extensively with 0.005 M K2HPO4 pH 7.2, 10% glycerol, 0.001 M DTT and 0.0005 M PMSF (contaminating intein is removed at this step), and the enzyme eluted with 0.3 M K2HPO4, pH 7.2, 10% glycerol, 0.001 M DTT and 0.0005 M PMSF. Fractions containing protein were pooled and dialyzed against 0.01 M Tris pH 8.0, 0.1 M NaCl, 0.001 M EDTA, 0.001 M DTT and 50% (v/v) glycerol, and stored at –20°C. The concentration of the enzyme was determined by a BioRad protein assay and its purity examined by SDS–PAGE. All preparations were 80% pure with the major contaminant being intein. All pol/prim preparations were also tested for activity in a monopolymerase DNA replication assay as described (17).
Purification of RPA
Recombinant human RPA was purified from transformed Escherichia coli as described (56). It was judged to be >95% pure.
Biotinylated DNA binding assays
Most assays were carried out in 40 μl of complete DNA replication buffer (0.03 M HEPES pH 8.0, 0.007 M MgCl2, 0.0005 M DTT, 0.04 M creatine phosphate, 0.004 M ATP, 0.0002 M CTP, UTP and GTP, 0.0001 M dATP, dGTP and dTTP, 0.00002 M dCTP, 50 μg/ml bovine serum albumin, 25 μg/ml phosphocreatine kinase). Each reaction contained 4.5 ng of biotinylated DNA fragment (–55 to 128 biotin or –64 to 128 biotin), 450 ng of T antigen and various amounts of purified topo I, pol/prim and/or RPA as described for each experiment. The assay in Figure 4 utilized minimal buffer (0.03 M HEPES pH 8.0, 0.007 M MgCl2, 0.0005 M DTT, 50 μg/ml bovine serum albumin). After 20 min at 37°C, or at the end of the incubation, 40 ng of pSK(+) DNA (Stratagene) was added to reduce nonspecific binding of topo I and RPA to the biotinylated origin DNA and incubation continued for an additional 5 min at 37°C. This treatment had no effect on the binding of these proteins to the DNA in the presence of T antigen. Glutaraldehyde was added to a final concentration of 0.06%, and 15 μl of a suspension of paramagnetic beads (Promega) previously washed in DNA wash buffer (0.01 M Tris pH 8.0, 0.15 M NaCl, 0.5% NP-40) was added. After 10 min at room temperature, the beads were pulled from solution with a magnet and washed three times with DNA wash buffer and once with Tris-buffered saline. The beads were then resuspended in 10 μl of 2% SDS and incubated for 15 min at 37 C. After removing the beads, 0.5 μl (for T antigen) or 2 μl (for topo I and RPA) was spotted on nitrocellulose membranes. Each protein was detected and quantitated by dot blots using PAb101 antibody (54) for T antigen, 8G6 antibody (15) for topo I and Ab-1 plus Ab-2 directed against RPA (Oncogene). Horseradish-peroxidase-conjugated anti-mouse antibody (Sigma) was then added followed by ChemiGlow (Alpha Innotech) substrate. Detection and quantitations of chemiluminescence were performed with a FluorChem 8800 (Alpha Innotech). Background binding of topo I or RPA to the DNA was determined by reactions performed without T antigen and these values were subtracted from those with T antigen. Typically, the background was 5% of the signal for topo I and 3–5% for RPA.
Figure 4. Effect of ATP S on the formation of intiation complex. Biotinylated origin DNA (–55 to 128) was incubated with T antigen (450 ng), topo I (40 ng), pol/prim (91 ng) and RPA (300 ng) in minimal buffer (no nucleotides) or minimal buffer containing ATP S. After 20 min at 37°C, the complexes were recovered by binding to streptavidin paramagnetic beads and the relative amounts of T antigen, topo I and RPA determined by quantitative dot blots.
Topo I: double-hexamer binding assay
This assay was performed as previously described (12). Binding assays contained 5 ng of origin-containing DNA fragment (HindIII–KpnI fragment from SV40 DNA or molar equivalent of other smaller fragments as described), 400 ng of immunoaffinity purified T antigen and various amounts of WT topo I in minimal buffer supplemented with 0.04 M creatine phosphate and 0.004 M ADP. After 20 min at 37°C, glutaraldehyde was added to 0.1% and incubation continued for an additional 10 min. The reactions were loaded onto a composite polyacrylamide–agarose gel as described (12). Protein–DNA complexes were transferred to nitrocellulose and the proteins on the membrane were detected by a western reaction with antibodies against T antigen or topo I. Detection was with ECL reagent (Amersham) followed by autoradiography. Quantitations were performed with a personal densitometer (Molecular Dynamics).
Results
Stoichiometry of T antigen and topo I in complex
As part of our efforts to understand the function and composition of the complex that initiates SV40 DNA replication, we investigated the stoichiometry between T antigen and topo I in association with origin DNA. To measure true binding between topo I and T antigen on the initiation complex, we used a double-hexamer-dependent binding assay as previously described by Gai et al. (12). T antigen and origin DNA were incubated with various amounts of topo I, and the DNA protein complexes were cross-linked with glutaraldehyde and then subjected to electrophoresis in a composite polyacrylamide–agarose gel followed by western blotting. The amounts of T antigen in double-hexamer bands (Fig. 2A) were estimated by comparison with dot blots using known amounts of the protein (Fig. 2A). Likewise, we estimated the amounts of topo I present in double-hexamer bands by comparison with dot blots with various amounts of topo I (Fig. 2B). The amounts of T antigen and topo I in each double-hexamer band were calculated directly from plots of signal strength versus amount of protein (Fig. 2C). The results show that, at maximum, the molar ratio of T antigen to topo I was 6:1. This corresponds to two molecules of topo I per double hexamer.
Figure 2. Determination of the stoichiometry between T antigen and topo I in complex with origin DNA. Purified T antigen was incubated with an origin DNA fragment in the presence of different amounts of purifed topo I as shown. After cross-linking, the complexes were subjected to gel electrophoresis followed by western blotting. (A) T antigen blot showing the positions on the gel of single (TH) and double (TDH) hexamers. At the bottom are dot blots with known amounts of T antigen. (B) Topo I blot of the same gel. The position of double hexamers is shown. Topo I dot blots with known amounts of protein are shown at the bottom. (C) Plots of signal strength versus amounts of T antigen or topo I in dot blots. The long arrows correspond to the amounts of T antigen or topo I present at 60 or 70 ng topo I in the reaction.
Influence of DNA on the recruitment of topo I
We then asked whether DNA influences the recruitment of topo I to the origin by investigating the effect of DNA sequences on both sides of the origin (see Fig. 1 for map). First, we constructed a series of DNA fragments containing the entire core origin plus additional sequences on the early side or additional sequences on the late side (Fig. 3). These fragments were then used in a double-hexamer binding assay as described above. Figure 3A (left half) demonstrates that sequences immediately neighboring the core origin on the late side had no apparent effect on the recruitment of topo I. When the DNA fragment was extended to base pair 128 (see Fig. 1), some reduction in topo I binding was detected (Fig. 3A and B), although we have no explanation for this. On the other hand, DNA sequences on the early side immediately adjacent to the core origin influenced topo I binding.
Figure 3. Effect of DNA length on the recruitment of topo I. (A) The left side of the figure tests the effect of DNA from the late side of origin and the right side tests the effect of DNA from the early side. The left side of each panel shows the topo I western blot and the right side shows the T antigen blot of the same gel. The DNA constructs used are indicated at the top of the figure. The positions of single and double hexamers are also indicated. (B and C) Quantitation of results. Results were plotted relative to the maximum signal for T antigen or topo I. Standard deviations were based on two trials. The relative topo I to T antigen ratios were also plotted.
Although T antigen has no preference among various fragments for assembling into double hexamers, topo I preferred to bind to DNA that contains the region between –32 and –55 (Fig. 3A and C). Therefore DNA length or the DNA on the early side of the origin appeared to stimulate topo I binding. To determine if T antigen binding site I was responsible for this effect, we compared two origin fragments, one with site I (–55 to 73) and a slightly smaller one with a 31 bp deletion of site I but starting further upstream (–72 to 73) (see Fig. 1). Both fragments were able to recruit topo I equally well (data not shown). This indicates that the additional DNA, but not site I itself, was responsible for this effect. In agreement with this, we found that double hexamers bound to the 64 bp core origin region recruited topo I poorly (data not shown).
Dynamics of assembly of the initiation complex
The experiments described above were performed in the absence of the other two initiation factors: DNA polymerase /primase and RPA. We set out to understand how the three cellular proteins participate in forming the initiation complex and, in particular, we were interested in determining the order in which these three proteins bound to the complex. We have previously hypothesized (2) that topo I may be the first cellular protein recruited to the complex based on the fact that its binding depended only on a fully formed double hexamer and not on other factors such as the presence of additional proteins or the hydrolysis of ATP (12). However, this has not been examined directly. The formation of T antigen double hexamers is known to occur in the presence of nonhydrolyzable ATP analogs (12,57), and we first showed here that RPA can associate with the initiation complex in the presence of the ATP S (Fig. 4), just as we demonstrated previously for topo I (12). Hence our previous assumption may well have been wrong.
To investigate the influence of each protein on the formation of initiation complexes, we added various amounts of each protein and determined the quantity of each protein bound to origin DNA. These experiments were performed with origin DNA that was biotinylated at one site. Various buffer conditions were tested and the one that gave the best signals for all proteins was a complete replication buffer (see Materials and Methods). Although this buffer can support DNA synthesis (all nucleotides including ATP are present), there was little DNA synthesis under these binding conditions primarily because the DNA that was used is a small fragment, not a circular plasmid, and as a consequence DNA synthesis efficiency was reduced 20- to 50-fold (data not shown). After the binding reactions, the DNA and any associated proteins were incubated with glutaraldehyde to cross-link them and then attached to streptavidin paramagnetic beads. Proteins bound to the beads were then detected and quantitated by dot blots using specific monoclonal antibodies. Control experiments demonstrated that glutaraldehyde cross-linking and SDS denaturation had minimal effects on the ability of antibodies to bind to their respective proteins (data not shown). These experiments were performed with fixed amounts of T antigen (450 ng) and origin DNA fragment (4.5 ng), representing 150-fold molar excess of T antigen to DNA. Under these conditions, 32 ng of T antigen bound maximally (100% value in Fig. 5A for example) corresponding to an 11:1 molar ratio of T antigen to DNA (assuming all the DNA bound to the beads), which is in close agreement with the expected 12:1 ratio for double hexamers bound to DNA.
Figure 5. Effects of topo I on assembly of initiation complex. (A) Biotinylated origin DNA (–64 to 128) was incubated with 450 ng T antigen, RPA (300 ng) and increasing amounts of topo I as shown. The relative amounts of each protein in the DNA bound fraction were determined by quantitative dot blots. These are plotted as a percentage of the maximum signal for each protein. The relative RPA to T antigen ratio is also plotted with the first point in the absence of topo I arbitrarily set at 1. (B) Same experiment but performed in the presence of DNA polymerase (91 ng). (C) Same experiment as in B except that RPA was missing. In addition to the T and topo I signals, the relative topo I to T antigen ratio is plotted with the maximum arbitrarily set at 1.
As a first test, we determined the relative amounts of T antigen, topo I and RPA bound to origin DNA when different amounts of topo I were added to the reaction but in the absence of DNA polymerase (Fig. 5A). We have observed previously that excess topo I interferes with the stability and function of double hexamers associated with origin DNA (12,13) and we wanted to see whether the same effect occurred in the presence of RPA. Figure 5A shows that topo I does have a detrimental effect on the stable association of T antigen with origin DNA under these conditions, as also illustrated in Figure 2. The concentration of topo I that inhibited the binding of T antigen appeared to be lower in this experiment than in the one shown in Figure 2. However, these two experiments used different DNAs, binding buffers and preparations of topo I, and are not directly comparable. The amounts of topo I bound to the complex in the binding assay (Fig. 5A) increased to a maximum when 20 ng of topo I were present and then decreased after that, most likely reflecting the decrease in the amounts of T antigen bound to DNA. At maximum, 3.6 ng of topo I were bound, representing a 1:6 molar ratio of topo I to T antigen in that sample. This is in excellent agreement with the results described above in Figure 2. The levels of RPA also increased, reaching a maximum at 20 ng of added topo I, and then decreased. At maximum, 4.7 ng of RPA were bound to the complex, corresponding to an 1:5 molar ratio of RPA to T antigen. This suggests that, like topo I, one molecule of RPA associates with each hexamer. When the levels of RPA bound to DNA were plotted as a ratio of the amounts of T antigen present, the curve paralleled that for topo I, indicating that topo I may have a role in recruiting RPA (Fig 5A).
We then investigated the effects of topo I on the formation of the complex in the presence of DNA pol/prim. Figure 5B shows that DNA polymerase has a protective effect against the disruptive activity of topo I. The amounts of T antigen stayed higher in the presence of larger amounts of topo I (compare the two T antigen curves in Fig. 5A and B). As a consequence, more topo I and RPA were recruited to the complex. The maximum amounts of topo I bound were reached at 40 ng of topo I added in the presence of DNA polymerase, as opposed to 20 ng in its absence. The presence of the DNA polymerase increased the maximum amount of topo I (and T antigen) bound by 3-fold in this experiment. Likewise, the amounts of RPA stayed high. As in Figure 5A, the RPA to T antigen ratio increased as more topo I was added, but in this case that number did not drop. These data suggest that topo I influences the amounts of RPA bound to the complex and that pol/prim may have a role in stabilizing the associated RPA in the presence of high concentrations of topo I.
When a similar experiment was performed by adding increasing amounts of topo I in the absence of RPA (Fig. 5C), we found that optimal amounts of topo I were present between 30 and 40 ng of topo I, slightly lower than when the experiment was performed in the presence of RPA (Fig. 5B). This indicates that RPA had a negligible effect on the recruitment of topo I except at high topo I concentrations where RPA appeared to be beneficial (compare Fig. 5B and C). RPA also had little effect on the binding of T antigen to the DNA (compare T antigen curves in Fig. 5B and C).
We then turned to a determination of how various amounts of DNA polymerase influence the association of various protein components with the origin. Figure 6A shows that as increasing amounts of DNA polymerase were added in the presence of fixed amounts of the other components, the complexes were stabilized dramatically compared with the absence of DNA polymerase. The amounts of T antigen and RPA reached saturation after 100 ng of DNA polymerase. However, the optimal amounts of topo I were present at 91 ng of DNA polymerase added and declined after that. This experiment confirms the protective effects of DNA polymerase on the formation of the initiation complex and also suggests that DNA polymerase modulates the binding of topo I to the complex. At low concentrations DNA polymerase stimulates topo I binding by stabilizing initiation complexes, but at high concentrations DNA polymerase is inhibitory. Similar results were obtained when the same experiment was performed in the absence of RPA (data not shown), indicating that RPA has no apparent role in the regulation of topo I binding by DNA polymerase or in the recruitment of the other proteins to the origin complex.
Figure 6. Effects of DNA polymerase on assembly of complex. (A) Biotinylated origin DNA (–55 to 128) was incubated with 450 ng T antigen, 300 ng RPA, 40 ng topo I and various amounts of pol/prim as shown. The relative T, topo I and RPA signals are plotted as is the relative topo I/T ratio (arbitrarily set at 1 in the absence of pol/prim). (B) Same experiment only topo I is missing. The relative RPA to T ratio is plotted.
In the absence of topo I (Fig. 6B), DNA polymerase had no major effect on the ability of T antigen or RPA to bind to the DNA, suggesting that its main role in assembly is to protect the complex from disruption by topo I. Repeated attempts to demonstrate that DNA polymerase was also part of the complex failed. At times, we could see a weak pol/prim signal, but it was difficult to quantitate and was not reproducible. The absence of a DNA polymerase signal was not due to technical problems since controls with known amounts of DNA polymerase (or the DNA polymerase in the binding reaction) worked well. The pol/prim signal was also not enhanced by the absence of RPA and/or topo I. In spite of the absence of detectable bound DNA polymerase, it was clear that the enzyme exhibited a positive effect on the formation and stability of the complex.
Subsequently, we investigated how various amounts of RPA changed the formation of the complex. These experiments were performed in the presence (Fig. 7A) and the absence (Fig. 7B) of DNA polymerase. RPA appeared to have no influence on the recruitment of T antigen or topo I to the complex whether or not DNA polymerase was present (in the presence of 40 ng of topo I). The topo I to T antigen ratio was unchanged at all RPA concentrations. In the absence of pol/prim (Fig. 7B), the RPA, T antigen and topo I signals were lower (by 4- to 5-fold) than in the presence of pol/prim (Fig. 7A), and this led to more scatter in the data. However, our conclusions were the same.
Figure 7. Effect of RPA on assembly of complex. (A) Biotinylated origin DNA (–64 to 128) was incubated with 450 ng T antigen, 40 ng topo I, 91 ng pol/prim and various amounts of RPA as shown. The relative T antigen, topo I and RPA signals are plotted, as is the relative topo I to T antigen ratio (arbitrarily set at 1 in the absence of RPA). (B) Same experiment except that pol/prim was missing.
The absence of DNA polymerase from origin DNA suggested that this enzyme was not a stable component of the initiation complex under these conditions. An alternative explanation is that the polymerase is present but cannot be detected because the epitope that the antibody recognizes is masked. Since the presence of DNA polymerase can easily be measured by its influence on the integrity of the complex, we distinguished between these two possibilities by investigating whether DNA polymerase present in a first incubation could stabilize the complexes from the disruptive effects of topo I in a second incubation without the polymerase. We first incubated T antigen with origin DNA in the presence or absence of DNA polymerase. After the complexes formed, we bound them to streptavidin beads and then resuspended the beads in binding buffer containing no additional protein, or in the presence of topo I or DNA polymerase, or with topo I plus DNA polymerase. Table 1 shows that the presence of DNA polymerase in the first incubation had essentially no effect on the disruption of the complexes by topo I in the second incubation. However, the presence of DNA polymerase in the second incubation did protect against the loss of T antigen from the DNA by topo I. This experiment suggests that DNA polymerase is not a stable component of the complex and needs to be present at all times in order to stabilize the assembly of the complex. Our interpretation is that it associates and dissociates frequently.
Table 1. Effect of DNA polymerase on stability of initiation complex
Discussion
Our results indicate that DNA polymerase is needed continually and at a certain concentration to allow the formation of maximum amounts of initiation complexes consisting of origin DNA, T antigen, RPA and topo I (Fig. 6A). It is clearly needed to relieve the topo I induced disruption of complexes. Also, DNA polymerase influences the amounts of topo I bound to the DNA. For these reasons, we propose that this enzyme is needed first to stabilize the complex and permit the subsequent association of the correct amounts of the other proteins. It appears, however, that under the conditions we used DNA polymerase is not itself a stable component of the complex since, if it were, it would stabilize against disruption by topo I when present in a first incubation (Table 1). Topo I enhances the recruitment of RPA either by directly binding to it or by upregulating T antigen’s ability to bind RPA (Fig. 5). We interpret this observation to mean that topo I is recruited second or that it comes in associated with RPA. RPA appears to have the least influence on the recruitment of the other components to the initiation complex, and we therefore propose that it could be recruited last.
The absence of detectable DNA polymerase from the complex was surprising in light of its known interactions with T antigen (5,8,41,42). The pol/prim dot blot signal (when we could detect it) was no more than 10% of that of RPA and topo I, implying an upper limit of less than one molecule of pol/prim for each of five DNA–protein complexes. However, T antigen–DNA polymerase binding is hard to measure and has not been characterized in the context of the functionally significant double hexamer. Based on our inability to detect significant amounts associated with T antigen–DNA complexes we infer that DNA polymerase must touch down on the template only for short periods of time. This is consistent with its ability to synthesize only short stretches of DNA (17,44). It is clear that binding can be measured under some conditions, but it appears from our results that binding is not stable under conditions that permit initiation of DNA replication. Another explanation is that binding of pol/prim is stabilized by elongation factors such as pol , PCNA and/or RF-C, but this has not been tested.
How topo I destabilizes T antigen double-hexamer complexes at the origin is not known. One possibility is that its binding to DNA directly causes it to dislodge T antigen. Another possibility is that when it binds to T antigen, T antigen loosens its grip with DNA through binding-induced structural changes. DNA polymerase relieves that inhibition either by binding directly to topo I or, more likely, by binding to T antigen and rendering T antigen less sensitive to disruption by topo I. Since excess pol/prim also downregulates the amounts of topo I that bind, it is clear that it is actively changing the make-up of the complex.
Likewise, topo I helps recruit RPA either by directly binding to it or by permitting the RPA binding site on T antigen to be more accessible. Recently (39), we have mapped the two topo I binding sites on T antigen and have shown that one of them is located between residues 83 and 160. This is just adjacent to the reported binding site on T antigen for RPA (residues 164–249) (48). Therefore it is possible that the binding of topo I to its site in the N-terminal domain alters the structure of that region to facilitate RPA binding. Pol/prim may also have a role in recruiting RPA especially at high topo I concentrations (Fig. 5B), although it appears that T antigen and topo I are primarily responsible for the binding of RPA. This is also evident from Figure 6B where pol/prim had no significant effect on RPA recruitment in the absence of topo I. The likely explanation is that DNA polymerase is mainly stabilizing the double-hexamer structure when topo I is present and therefore there are sufficient T antigen–topo I complexes to recruit RPA directly.
The stoichiometry between topo I and T antigen in the double-hexamer structure was found to be approximately one molecule of topo I to six molecules of T antigen (Fig. 2). These experiments were initially performed in the absence of the other proteins. However, the same stoichiometry was obtained in the presence of RPA (Fig. 5A) and RPA plus DNA polymerase (Fig. 5B). Although we did not do the RPA stoichiometric analysis as rigorously, it appeared that this protein was also present in the same molar ratio.
The influence of DNA from the early side of the EP region on the recruitment of topo I to the complex suggests that DNA has a direct role in binding and bringing topo I to the initiation complex. This effect is not dependent on T antigen binding site I and it is possible that any extra DNA may facilitate topo I recruitment. At this point, it is not known whether the additional DNA must be on the early side or if it can be on the late side as well since the constructs with varying amounts of DNA from the late side were all larger than the –32 to +41 origin fragment. We have shown that double hexamers assembled over the 64 bp core origin are also able to recruit topo I, but do so weakly with significantly less efficiency than longer DNAs (data not shown). Since T antigen is thought to cover and protect the entire core origin, this indicates that topo I may be recruited to the complex through interactions with T antigen as well as the DNA.
Our working model is that pol/prim is the first cellular protein to interact with the T antigen double hexamer, but this enzyme is only transiently or weakly bound. Pol/prim is required continually to prevent topo I from disrupting the complex. Topo I, in turn, associates through interactions with T antigen and with the DNA. In the complex, one molecule of topo I is bound to each hexamer. T antigen and topo I, perhaps working together, are responsible for recruiting RPA to the initiation complex (one molecule per hexamer). RPA has no major effect on the presence of the other components in the complex, implying that it could be recruited last.
In contrast to our results suggesting that recruitment of RPA occurs after pol/prim, a number of investigators (58–60) have found that in the Xenopus oocyte system, as well as in Saccharomyces, loading of pol/prim onto chromatin is dependent on prior binding by RPA. There are several obvious differences between the SV40 and the cellular DNA replication systems that could account for this disparity. First, the viral system depends on T antigen for helicase activity whereas the cellular system relies on the MCM proteins. The first provides for unregulated multiple firing of the origin; the second is regulated and fires only once per cell cycle, and this basic functional difference may require a different loading order. Second, RPA, pol/prim and topo I can bind directly to T antigen in the SV40 system; it is not known if these proteins bind to MCM directly and it is quite possible that they are recruited by binding to any of the other multiple components of the pre-replication complex. Third, although a less likely possible explanation, the SV40 DNA used here is not associated with histones in chromatin unlike cellular chromosomal DNA. Any or all of these explanations could account for this difference.
ACKNOWLEDGEMENTS
This work was supported by grant CA36118 from the National Cancer Institute to D.T.S.
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*To whom correspondence should be addressed. Tel: +1 302 831 8547; Fax: +1 302 831 2281; Email: dsimmons@udel.edu
ABSTRACT
The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase /primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.
Introduction
Replication of simian virus 40 (SV40) DNA begins when the virally coded T antigen binds to the SV40 origin and forms a double-hexameric structure . Subsequently, three cellular proteins, replication protein A (RPA) (4–7), DNA polymerase /primase (pol/prim) (8–11) and topoisomerase I (topo I) (12–15), associate with T antigen to generate an initiation complex. After initiation, the majority of DNA synthesis on both leading and lagging strands occurs in response to additional cellular proteins such as polymerase , PCNA and RF-C (16–18). In all, 11 cellular proteins are utilized to fully replicate the virus DNA (19–25). Although all of these proteins have been purified and studied to investigate their role in replication, there is only marginal understanding of the dynamics of the replication process. Surprisingly, there is little information about how the initiation complex itself assembles and how the three cellular proteins work with the T antigen double hexamer to initiate the synthesis of RNA primers and to extend these primers with DNA.
The assembly of the T antigen double hexamer is fairly well understood. A single monomer is believed to bind to pentanucleotide 1 at the origin (see Fig. 1) and associate with others to form a single hexamer (26–29). In addition to pentanucleotide 1, flanking sequences consisting of the early palindrome (EP) region (Fig. 1) are necessary for formation of the first hexamer (27). A second hexamer then forms from individual monomers over pentanucleotide 3 (26,28). These three origin elements (pentanucleotides 1 and 3 and EP) constitute one assembly unit for the construction of a double hexamer (26). A second assembly unit consisting of pentanucleotides 2 and 4 and the AT track can also support formation of a double hexamer, but does so less efficiently. When a double hexamer forms over the origin, 8 bp of the DNA at the EP region are melted and the DNA at the AT track is untwisted (30–35). These two structural changes together are often referred as ‘structural distortion’. Structural distortion is believed to precede the melting of the origin by T antigen’s helicase activity. The mechanism by which this occurs in under intensive investigation in several laboratories and is believed to be accompanied by extensive structural changes in T antigen as well as in the DNA (36).
Figure 1. Sequence of the SV40 origin. The core origin is indicated between the two double triangles and consists of the EP region, site II and the AT track. Arrows within site II (shown in red) represent each of the four GAGGC pentanucleotides that T antigen recognizes. Arrows within the EP region represent an imperfect palindromic sequence. T antigen binding site I (highlighted in green) is shown on the early side of the core origin. Each of its two pentanucleotides is indicated as an arrow. The sequence between the single blue triangles is deleted in pSKORI.
Only recently has it been recognized that topo I is an integral component of the initiation complex (12,15). This enzyme nicks and religates DNA in order to relieve torsional strain ahead of the replication fork, and there is increasing evidence that it functions in combination with T antigen as a gyrase to unwind and relax DNA (12–15,37,38). Very recently, nucleolin has also been implicated as an initiation factor that facilitates assembly and activity of a gyrase complex (38). Although there is no evidence that nucleolin is needed in vitro for DNA replication, it may well be needed in infected cells. Topo I forms a specific interaction with double hexamers and does not bind well to smaller oligomerized forms of T antigen (12). By mapping the sites of interaction between T antigen and topo I, we have recently proposed that topo I binds to the ends of the double-hexamer complex (39). In this orientation, we hypothesize that the nicking and religation activity of topo I is coupled to the unwinding activity of T antigen to efficiently propel the replication fork.
Pol/prim consists of four subunits, of which one (p180) is the polymerase catalytic subunit, one (p68) is a structural unit and the other two (p58 and p48) are components of the primase (40). T antigen binds to the p180 subunit (41) and stoichiometric analysis predicts a ratio of one pol/prim molecule per T antigen hexamer (42). The primase synthesizes 8–10 nt stretches of RNA starting at sites with a consensus sequence of 5'-TTN-3' (43). Pol/prim then extends these RNA primers with 35 nt of DNA (17,44). The enzyme is not very processive and readily falls off its template (40). Further DNA synthesis requires elongation factors DNA polymerase , PCNA and RF-C (6,16,17).
RPA is a three-subunit protein that coats and protects exposed single-stranded regions of DNA at replication forks (45). RPA also has the added function to stimulate pol/prim to lay down RNA primers (46). The largest subunit (p70) has single-stranded DNA binding activity as well as the ability to bind T antigen (47). The binding site for RPA on T antigen has been mapped to residues 164–249 (48). RPA can also bind directly to pol/prim apparently through interactions involving all three RPA subunits (5,49,50).
DNA synthesis can take place in vitro using the so-called ‘monopolymerase system’, which consists of plasmid DNA containing the SV40 origin, T antigen and all three cellular proteins described above (9,17,51,52). Under these conditions, a heterogenous population of DNA molecules is synthesized ranging in size from a few nucleotides to several thousand. Optimal synthesis of DNA is dependent on all four proteins.
In this study, we determined the stoichiometry between T antigen and topo I and evaluated the dynamics of assembly of the initiation complex. We show that DNA polymerase appears to be the first cellular protein to be required for the formation of the initiation complex. However, this enzyme cannot be detected in the complex, although its presence is needed for T antigen double hexamers to remain associated with origin DNA in the presence of topo I and for subsequent proteins such as topo I itself and RPA to bind optimally.
Materials and Methods
Construction of origin DNA fragments
All origin-containing fragments were generated by PCR amplification of the SV40 origin from pSKori (13). Appropriate primers 20 nt long were designed to amplify specific regions of the origin to make the fragments used in this study. These corresponded in sequence to that shown in Figure 1 starting with each arrow. A fragment missing T antigen binding site I was made by PCR from pSKORI, which is identical to pSKori except for a deletion of 31 bp as shown in Figure 1. One PCR primer for this fragment started at –72 and extended beyond the deletion to nucleotide –24. Biotinylated DNA was made by PCR using a ‘down’-biotinylated oligonucleotide whose 5' end was modified with biotin and which corresponds to residue +128 (Fig. 1) and an ‘up’ primer with 5' end at either –64 or –55. DNA fragments were purified by agarose gel electrophoresis.
Purification of T antigen
WT T antigen was purified from baculovirus-infected insect cells by immunoaffinity chromatography as described previously (53) using PAb101 antibody (54). All preparations were >90% pure.
Purification of topo I
WT topo I was purified by standard chromatography from extracts of baculovirus-infected Sf9 cells as described previously (55). Purity was >90%.
Purification of pol/prim
Pol/prim was purified by a novel procedure that permitted the preparation of highly pure and active enzyme in two steps. The procedure was based on the Impact-CN system (New England Biolabs) where one of the subunits was tagged with a modified form of intein. First, the cDNA for the p58 primase subunit of the enzyme was generated by PCR from baculovirus DNA expressing that subunit (the virus was a gift from Dr Ellen Fanning). The PCR primers were designed according to the manufacturer’s directions for insertion into pTYB3 (NEB). The PCR-generated cDNA was digested with XbaI and SapI and ligated into gel-purified linear pTYB3 digested with the same enzymes. Clones were screened for the presence of the insert in the correct orientation by restriction digestion with XbaI and PstI, and potential clones were subjected to DNA sequencing. The DNA of clones with the correct sequence was digested with the same two enzymes and the smaller fragment was ligated into the equivalent sites of p1393 (Pharmingen) baculovirus transfer vector. Recombinant baculovirus was made by cotransfection into Sf9 insect cells as described by the manufacturer (Pharmingen). Expressing virus was screened by immunofluorescence using an antibody directed against the primase subunits (a kind gift from H. P. Nasheuer).
To generate pol/prim, High 5 insect cells were infected with the recombinant virus expressing the intein tagged p58 primase subunit and co-infected with recombinant baculoviruses expressing each of the other three untagged pol/prim subunits (kind gifts from Dr Ellen Fanning and Dr T. S. Wang). At 60 h post-infection, the cells were lysed by dounce homogenization in 0.15 M Tris pH 7.0, 0.15 M NaCl, 0.001 M EDTA, 10% glycerol, 0.5% NP40 and 0.001 M phenyl-methylsulfonyl fluoride (PMSF) and the clarified lysate was incubated with chitin beads. After 1 h at 4°C, the beads were centrifuged and washed twice with 0.05 M Tris pH 7.0, 0.5 M NaCl, 0.001 M EDTA, 10% glycerol and 1% NP40, and twice with the same buffer minus the NP40. The beads were transferred to a small column and two column volumes of cleavage buffer were quickly passed over the column. More cleavage buffer was added and the column was left stoppered in the cold room overnight. The next day, the eluate was recovered into several fractions. Fractions containing protein were pooled and the pol/prim was further purified and concentrated over a small (0.5 ml) column containing hydroxylapatite equilibrated in cleavage buffer lacking DTT. The column was washed extensively with 0.005 M K2HPO4 pH 7.2, 10% glycerol, 0.001 M DTT and 0.0005 M PMSF (contaminating intein is removed at this step), and the enzyme eluted with 0.3 M K2HPO4, pH 7.2, 10% glycerol, 0.001 M DTT and 0.0005 M PMSF. Fractions containing protein were pooled and dialyzed against 0.01 M Tris pH 8.0, 0.1 M NaCl, 0.001 M EDTA, 0.001 M DTT and 50% (v/v) glycerol, and stored at –20°C. The concentration of the enzyme was determined by a BioRad protein assay and its purity examined by SDS–PAGE. All preparations were 80% pure with the major contaminant being intein. All pol/prim preparations were also tested for activity in a monopolymerase DNA replication assay as described (17).
Purification of RPA
Recombinant human RPA was purified from transformed Escherichia coli as described (56). It was judged to be >95% pure.
Biotinylated DNA binding assays
Most assays were carried out in 40 μl of complete DNA replication buffer (0.03 M HEPES pH 8.0, 0.007 M MgCl2, 0.0005 M DTT, 0.04 M creatine phosphate, 0.004 M ATP, 0.0002 M CTP, UTP and GTP, 0.0001 M dATP, dGTP and dTTP, 0.00002 M dCTP, 50 μg/ml bovine serum albumin, 25 μg/ml phosphocreatine kinase). Each reaction contained 4.5 ng of biotinylated DNA fragment (–55 to 128 biotin or –64 to 128 biotin), 450 ng of T antigen and various amounts of purified topo I, pol/prim and/or RPA as described for each experiment. The assay in Figure 4 utilized minimal buffer (0.03 M HEPES pH 8.0, 0.007 M MgCl2, 0.0005 M DTT, 50 μg/ml bovine serum albumin). After 20 min at 37°C, or at the end of the incubation, 40 ng of pSK(+) DNA (Stratagene) was added to reduce nonspecific binding of topo I and RPA to the biotinylated origin DNA and incubation continued for an additional 5 min at 37°C. This treatment had no effect on the binding of these proteins to the DNA in the presence of T antigen. Glutaraldehyde was added to a final concentration of 0.06%, and 15 μl of a suspension of paramagnetic beads (Promega) previously washed in DNA wash buffer (0.01 M Tris pH 8.0, 0.15 M NaCl, 0.5% NP-40) was added. After 10 min at room temperature, the beads were pulled from solution with a magnet and washed three times with DNA wash buffer and once with Tris-buffered saline. The beads were then resuspended in 10 μl of 2% SDS and incubated for 15 min at 37 C. After removing the beads, 0.5 μl (for T antigen) or 2 μl (for topo I and RPA) was spotted on nitrocellulose membranes. Each protein was detected and quantitated by dot blots using PAb101 antibody (54) for T antigen, 8G6 antibody (15) for topo I and Ab-1 plus Ab-2 directed against RPA (Oncogene). Horseradish-peroxidase-conjugated anti-mouse antibody (Sigma) was then added followed by ChemiGlow (Alpha Innotech) substrate. Detection and quantitations of chemiluminescence were performed with a FluorChem 8800 (Alpha Innotech). Background binding of topo I or RPA to the DNA was determined by reactions performed without T antigen and these values were subtracted from those with T antigen. Typically, the background was 5% of the signal for topo I and 3–5% for RPA.
Figure 4. Effect of ATP S on the formation of intiation complex. Biotinylated origin DNA (–55 to 128) was incubated with T antigen (450 ng), topo I (40 ng), pol/prim (91 ng) and RPA (300 ng) in minimal buffer (no nucleotides) or minimal buffer containing ATP S. After 20 min at 37°C, the complexes were recovered by binding to streptavidin paramagnetic beads and the relative amounts of T antigen, topo I and RPA determined by quantitative dot blots.
Topo I: double-hexamer binding assay
This assay was performed as previously described (12). Binding assays contained 5 ng of origin-containing DNA fragment (HindIII–KpnI fragment from SV40 DNA or molar equivalent of other smaller fragments as described), 400 ng of immunoaffinity purified T antigen and various amounts of WT topo I in minimal buffer supplemented with 0.04 M creatine phosphate and 0.004 M ADP. After 20 min at 37°C, glutaraldehyde was added to 0.1% and incubation continued for an additional 10 min. The reactions were loaded onto a composite polyacrylamide–agarose gel as described (12). Protein–DNA complexes were transferred to nitrocellulose and the proteins on the membrane were detected by a western reaction with antibodies against T antigen or topo I. Detection was with ECL reagent (Amersham) followed by autoradiography. Quantitations were performed with a personal densitometer (Molecular Dynamics).
Results
Stoichiometry of T antigen and topo I in complex
As part of our efforts to understand the function and composition of the complex that initiates SV40 DNA replication, we investigated the stoichiometry between T antigen and topo I in association with origin DNA. To measure true binding between topo I and T antigen on the initiation complex, we used a double-hexamer-dependent binding assay as previously described by Gai et al. (12). T antigen and origin DNA were incubated with various amounts of topo I, and the DNA protein complexes were cross-linked with glutaraldehyde and then subjected to electrophoresis in a composite polyacrylamide–agarose gel followed by western blotting. The amounts of T antigen in double-hexamer bands (Fig. 2A) were estimated by comparison with dot blots using known amounts of the protein (Fig. 2A). Likewise, we estimated the amounts of topo I present in double-hexamer bands by comparison with dot blots with various amounts of topo I (Fig. 2B). The amounts of T antigen and topo I in each double-hexamer band were calculated directly from plots of signal strength versus amount of protein (Fig. 2C). The results show that, at maximum, the molar ratio of T antigen to topo I was 6:1. This corresponds to two molecules of topo I per double hexamer.
Figure 2. Determination of the stoichiometry between T antigen and topo I in complex with origin DNA. Purified T antigen was incubated with an origin DNA fragment in the presence of different amounts of purifed topo I as shown. After cross-linking, the complexes were subjected to gel electrophoresis followed by western blotting. (A) T antigen blot showing the positions on the gel of single (TH) and double (TDH) hexamers. At the bottom are dot blots with known amounts of T antigen. (B) Topo I blot of the same gel. The position of double hexamers is shown. Topo I dot blots with known amounts of protein are shown at the bottom. (C) Plots of signal strength versus amounts of T antigen or topo I in dot blots. The long arrows correspond to the amounts of T antigen or topo I present at 60 or 70 ng topo I in the reaction.
Influence of DNA on the recruitment of topo I
We then asked whether DNA influences the recruitment of topo I to the origin by investigating the effect of DNA sequences on both sides of the origin (see Fig. 1 for map). First, we constructed a series of DNA fragments containing the entire core origin plus additional sequences on the early side or additional sequences on the late side (Fig. 3). These fragments were then used in a double-hexamer binding assay as described above. Figure 3A (left half) demonstrates that sequences immediately neighboring the core origin on the late side had no apparent effect on the recruitment of topo I. When the DNA fragment was extended to base pair 128 (see Fig. 1), some reduction in topo I binding was detected (Fig. 3A and B), although we have no explanation for this. On the other hand, DNA sequences on the early side immediately adjacent to the core origin influenced topo I binding.
Figure 3. Effect of DNA length on the recruitment of topo I. (A) The left side of the figure tests the effect of DNA from the late side of origin and the right side tests the effect of DNA from the early side. The left side of each panel shows the topo I western blot and the right side shows the T antigen blot of the same gel. The DNA constructs used are indicated at the top of the figure. The positions of single and double hexamers are also indicated. (B and C) Quantitation of results. Results were plotted relative to the maximum signal for T antigen or topo I. Standard deviations were based on two trials. The relative topo I to T antigen ratios were also plotted.
Although T antigen has no preference among various fragments for assembling into double hexamers, topo I preferred to bind to DNA that contains the region between –32 and –55 (Fig. 3A and C). Therefore DNA length or the DNA on the early side of the origin appeared to stimulate topo I binding. To determine if T antigen binding site I was responsible for this effect, we compared two origin fragments, one with site I (–55 to 73) and a slightly smaller one with a 31 bp deletion of site I but starting further upstream (–72 to 73) (see Fig. 1). Both fragments were able to recruit topo I equally well (data not shown). This indicates that the additional DNA, but not site I itself, was responsible for this effect. In agreement with this, we found that double hexamers bound to the 64 bp core origin region recruited topo I poorly (data not shown).
Dynamics of assembly of the initiation complex
The experiments described above were performed in the absence of the other two initiation factors: DNA polymerase /primase and RPA. We set out to understand how the three cellular proteins participate in forming the initiation complex and, in particular, we were interested in determining the order in which these three proteins bound to the complex. We have previously hypothesized (2) that topo I may be the first cellular protein recruited to the complex based on the fact that its binding depended only on a fully formed double hexamer and not on other factors such as the presence of additional proteins or the hydrolysis of ATP (12). However, this has not been examined directly. The formation of T antigen double hexamers is known to occur in the presence of nonhydrolyzable ATP analogs (12,57), and we first showed here that RPA can associate with the initiation complex in the presence of the ATP S (Fig. 4), just as we demonstrated previously for topo I (12). Hence our previous assumption may well have been wrong.
To investigate the influence of each protein on the formation of initiation complexes, we added various amounts of each protein and determined the quantity of each protein bound to origin DNA. These experiments were performed with origin DNA that was biotinylated at one site. Various buffer conditions were tested and the one that gave the best signals for all proteins was a complete replication buffer (see Materials and Methods). Although this buffer can support DNA synthesis (all nucleotides including ATP are present), there was little DNA synthesis under these binding conditions primarily because the DNA that was used is a small fragment, not a circular plasmid, and as a consequence DNA synthesis efficiency was reduced 20- to 50-fold (data not shown). After the binding reactions, the DNA and any associated proteins were incubated with glutaraldehyde to cross-link them and then attached to streptavidin paramagnetic beads. Proteins bound to the beads were then detected and quantitated by dot blots using specific monoclonal antibodies. Control experiments demonstrated that glutaraldehyde cross-linking and SDS denaturation had minimal effects on the ability of antibodies to bind to their respective proteins (data not shown). These experiments were performed with fixed amounts of T antigen (450 ng) and origin DNA fragment (4.5 ng), representing 150-fold molar excess of T antigen to DNA. Under these conditions, 32 ng of T antigen bound maximally (100% value in Fig. 5A for example) corresponding to an 11:1 molar ratio of T antigen to DNA (assuming all the DNA bound to the beads), which is in close agreement with the expected 12:1 ratio for double hexamers bound to DNA.
Figure 5. Effects of topo I on assembly of initiation complex. (A) Biotinylated origin DNA (–64 to 128) was incubated with 450 ng T antigen, RPA (300 ng) and increasing amounts of topo I as shown. The relative amounts of each protein in the DNA bound fraction were determined by quantitative dot blots. These are plotted as a percentage of the maximum signal for each protein. The relative RPA to T antigen ratio is also plotted with the first point in the absence of topo I arbitrarily set at 1. (B) Same experiment but performed in the presence of DNA polymerase (91 ng). (C) Same experiment as in B except that RPA was missing. In addition to the T and topo I signals, the relative topo I to T antigen ratio is plotted with the maximum arbitrarily set at 1.
As a first test, we determined the relative amounts of T antigen, topo I and RPA bound to origin DNA when different amounts of topo I were added to the reaction but in the absence of DNA polymerase (Fig. 5A). We have observed previously that excess topo I interferes with the stability and function of double hexamers associated with origin DNA (12,13) and we wanted to see whether the same effect occurred in the presence of RPA. Figure 5A shows that topo I does have a detrimental effect on the stable association of T antigen with origin DNA under these conditions, as also illustrated in Figure 2. The concentration of topo I that inhibited the binding of T antigen appeared to be lower in this experiment than in the one shown in Figure 2. However, these two experiments used different DNAs, binding buffers and preparations of topo I, and are not directly comparable. The amounts of topo I bound to the complex in the binding assay (Fig. 5A) increased to a maximum when 20 ng of topo I were present and then decreased after that, most likely reflecting the decrease in the amounts of T antigen bound to DNA. At maximum, 3.6 ng of topo I were bound, representing a 1:6 molar ratio of topo I to T antigen in that sample. This is in excellent agreement with the results described above in Figure 2. The levels of RPA also increased, reaching a maximum at 20 ng of added topo I, and then decreased. At maximum, 4.7 ng of RPA were bound to the complex, corresponding to an 1:5 molar ratio of RPA to T antigen. This suggests that, like topo I, one molecule of RPA associates with each hexamer. When the levels of RPA bound to DNA were plotted as a ratio of the amounts of T antigen present, the curve paralleled that for topo I, indicating that topo I may have a role in recruiting RPA (Fig 5A).
We then investigated the effects of topo I on the formation of the complex in the presence of DNA pol/prim. Figure 5B shows that DNA polymerase has a protective effect against the disruptive activity of topo I. The amounts of T antigen stayed higher in the presence of larger amounts of topo I (compare the two T antigen curves in Fig. 5A and B). As a consequence, more topo I and RPA were recruited to the complex. The maximum amounts of topo I bound were reached at 40 ng of topo I added in the presence of DNA polymerase, as opposed to 20 ng in its absence. The presence of the DNA polymerase increased the maximum amount of topo I (and T antigen) bound by 3-fold in this experiment. Likewise, the amounts of RPA stayed high. As in Figure 5A, the RPA to T antigen ratio increased as more topo I was added, but in this case that number did not drop. These data suggest that topo I influences the amounts of RPA bound to the complex and that pol/prim may have a role in stabilizing the associated RPA in the presence of high concentrations of topo I.
When a similar experiment was performed by adding increasing amounts of topo I in the absence of RPA (Fig. 5C), we found that optimal amounts of topo I were present between 30 and 40 ng of topo I, slightly lower than when the experiment was performed in the presence of RPA (Fig. 5B). This indicates that RPA had a negligible effect on the recruitment of topo I except at high topo I concentrations where RPA appeared to be beneficial (compare Fig. 5B and C). RPA also had little effect on the binding of T antigen to the DNA (compare T antigen curves in Fig. 5B and C).
We then turned to a determination of how various amounts of DNA polymerase influence the association of various protein components with the origin. Figure 6A shows that as increasing amounts of DNA polymerase were added in the presence of fixed amounts of the other components, the complexes were stabilized dramatically compared with the absence of DNA polymerase. The amounts of T antigen and RPA reached saturation after 100 ng of DNA polymerase. However, the optimal amounts of topo I were present at 91 ng of DNA polymerase added and declined after that. This experiment confirms the protective effects of DNA polymerase on the formation of the initiation complex and also suggests that DNA polymerase modulates the binding of topo I to the complex. At low concentrations DNA polymerase stimulates topo I binding by stabilizing initiation complexes, but at high concentrations DNA polymerase is inhibitory. Similar results were obtained when the same experiment was performed in the absence of RPA (data not shown), indicating that RPA has no apparent role in the regulation of topo I binding by DNA polymerase or in the recruitment of the other proteins to the origin complex.
Figure 6. Effects of DNA polymerase on assembly of complex. (A) Biotinylated origin DNA (–55 to 128) was incubated with 450 ng T antigen, 300 ng RPA, 40 ng topo I and various amounts of pol/prim as shown. The relative T, topo I and RPA signals are plotted as is the relative topo I/T ratio (arbitrarily set at 1 in the absence of pol/prim). (B) Same experiment only topo I is missing. The relative RPA to T ratio is plotted.
In the absence of topo I (Fig. 6B), DNA polymerase had no major effect on the ability of T antigen or RPA to bind to the DNA, suggesting that its main role in assembly is to protect the complex from disruption by topo I. Repeated attempts to demonstrate that DNA polymerase was also part of the complex failed. At times, we could see a weak pol/prim signal, but it was difficult to quantitate and was not reproducible. The absence of a DNA polymerase signal was not due to technical problems since controls with known amounts of DNA polymerase (or the DNA polymerase in the binding reaction) worked well. The pol/prim signal was also not enhanced by the absence of RPA and/or topo I. In spite of the absence of detectable bound DNA polymerase, it was clear that the enzyme exhibited a positive effect on the formation and stability of the complex.
Subsequently, we investigated how various amounts of RPA changed the formation of the complex. These experiments were performed in the presence (Fig. 7A) and the absence (Fig. 7B) of DNA polymerase. RPA appeared to have no influence on the recruitment of T antigen or topo I to the complex whether or not DNA polymerase was present (in the presence of 40 ng of topo I). The topo I to T antigen ratio was unchanged at all RPA concentrations. In the absence of pol/prim (Fig. 7B), the RPA, T antigen and topo I signals were lower (by 4- to 5-fold) than in the presence of pol/prim (Fig. 7A), and this led to more scatter in the data. However, our conclusions were the same.
Figure 7. Effect of RPA on assembly of complex. (A) Biotinylated origin DNA (–64 to 128) was incubated with 450 ng T antigen, 40 ng topo I, 91 ng pol/prim and various amounts of RPA as shown. The relative T antigen, topo I and RPA signals are plotted, as is the relative topo I to T antigen ratio (arbitrarily set at 1 in the absence of RPA). (B) Same experiment except that pol/prim was missing.
The absence of DNA polymerase from origin DNA suggested that this enzyme was not a stable component of the initiation complex under these conditions. An alternative explanation is that the polymerase is present but cannot be detected because the epitope that the antibody recognizes is masked. Since the presence of DNA polymerase can easily be measured by its influence on the integrity of the complex, we distinguished between these two possibilities by investigating whether DNA polymerase present in a first incubation could stabilize the complexes from the disruptive effects of topo I in a second incubation without the polymerase. We first incubated T antigen with origin DNA in the presence or absence of DNA polymerase. After the complexes formed, we bound them to streptavidin beads and then resuspended the beads in binding buffer containing no additional protein, or in the presence of topo I or DNA polymerase, or with topo I plus DNA polymerase. Table 1 shows that the presence of DNA polymerase in the first incubation had essentially no effect on the disruption of the complexes by topo I in the second incubation. However, the presence of DNA polymerase in the second incubation did protect against the loss of T antigen from the DNA by topo I. This experiment suggests that DNA polymerase is not a stable component of the complex and needs to be present at all times in order to stabilize the assembly of the complex. Our interpretation is that it associates and dissociates frequently.
Table 1. Effect of DNA polymerase on stability of initiation complex
Discussion
Our results indicate that DNA polymerase is needed continually and at a certain concentration to allow the formation of maximum amounts of initiation complexes consisting of origin DNA, T antigen, RPA and topo I (Fig. 6A). It is clearly needed to relieve the topo I induced disruption of complexes. Also, DNA polymerase influences the amounts of topo I bound to the DNA. For these reasons, we propose that this enzyme is needed first to stabilize the complex and permit the subsequent association of the correct amounts of the other proteins. It appears, however, that under the conditions we used DNA polymerase is not itself a stable component of the complex since, if it were, it would stabilize against disruption by topo I when present in a first incubation (Table 1). Topo I enhances the recruitment of RPA either by directly binding to it or by upregulating T antigen’s ability to bind RPA (Fig. 5). We interpret this observation to mean that topo I is recruited second or that it comes in associated with RPA. RPA appears to have the least influence on the recruitment of the other components to the initiation complex, and we therefore propose that it could be recruited last.
The absence of detectable DNA polymerase from the complex was surprising in light of its known interactions with T antigen (5,8,41,42). The pol/prim dot blot signal (when we could detect it) was no more than 10% of that of RPA and topo I, implying an upper limit of less than one molecule of pol/prim for each of five DNA–protein complexes. However, T antigen–DNA polymerase binding is hard to measure and has not been characterized in the context of the functionally significant double hexamer. Based on our inability to detect significant amounts associated with T antigen–DNA complexes we infer that DNA polymerase must touch down on the template only for short periods of time. This is consistent with its ability to synthesize only short stretches of DNA (17,44). It is clear that binding can be measured under some conditions, but it appears from our results that binding is not stable under conditions that permit initiation of DNA replication. Another explanation is that binding of pol/prim is stabilized by elongation factors such as pol , PCNA and/or RF-C, but this has not been tested.
How topo I destabilizes T antigen double-hexamer complexes at the origin is not known. One possibility is that its binding to DNA directly causes it to dislodge T antigen. Another possibility is that when it binds to T antigen, T antigen loosens its grip with DNA through binding-induced structural changes. DNA polymerase relieves that inhibition either by binding directly to topo I or, more likely, by binding to T antigen and rendering T antigen less sensitive to disruption by topo I. Since excess pol/prim also downregulates the amounts of topo I that bind, it is clear that it is actively changing the make-up of the complex.
Likewise, topo I helps recruit RPA either by directly binding to it or by permitting the RPA binding site on T antigen to be more accessible. Recently (39), we have mapped the two topo I binding sites on T antigen and have shown that one of them is located between residues 83 and 160. This is just adjacent to the reported binding site on T antigen for RPA (residues 164–249) (48). Therefore it is possible that the binding of topo I to its site in the N-terminal domain alters the structure of that region to facilitate RPA binding. Pol/prim may also have a role in recruiting RPA especially at high topo I concentrations (Fig. 5B), although it appears that T antigen and topo I are primarily responsible for the binding of RPA. This is also evident from Figure 6B where pol/prim had no significant effect on RPA recruitment in the absence of topo I. The likely explanation is that DNA polymerase is mainly stabilizing the double-hexamer structure when topo I is present and therefore there are sufficient T antigen–topo I complexes to recruit RPA directly.
The stoichiometry between topo I and T antigen in the double-hexamer structure was found to be approximately one molecule of topo I to six molecules of T antigen (Fig. 2). These experiments were initially performed in the absence of the other proteins. However, the same stoichiometry was obtained in the presence of RPA (Fig. 5A) and RPA plus DNA polymerase (Fig. 5B). Although we did not do the RPA stoichiometric analysis as rigorously, it appeared that this protein was also present in the same molar ratio.
The influence of DNA from the early side of the EP region on the recruitment of topo I to the complex suggests that DNA has a direct role in binding and bringing topo I to the initiation complex. This effect is not dependent on T antigen binding site I and it is possible that any extra DNA may facilitate topo I recruitment. At this point, it is not known whether the additional DNA must be on the early side or if it can be on the late side as well since the constructs with varying amounts of DNA from the late side were all larger than the –32 to +41 origin fragment. We have shown that double hexamers assembled over the 64 bp core origin are also able to recruit topo I, but do so weakly with significantly less efficiency than longer DNAs (data not shown). Since T antigen is thought to cover and protect the entire core origin, this indicates that topo I may be recruited to the complex through interactions with T antigen as well as the DNA.
Our working model is that pol/prim is the first cellular protein to interact with the T antigen double hexamer, but this enzyme is only transiently or weakly bound. Pol/prim is required continually to prevent topo I from disrupting the complex. Topo I, in turn, associates through interactions with T antigen and with the DNA. In the complex, one molecule of topo I is bound to each hexamer. T antigen and topo I, perhaps working together, are responsible for recruiting RPA to the initiation complex (one molecule per hexamer). RPA has no major effect on the presence of the other components in the complex, implying that it could be recruited last.
In contrast to our results suggesting that recruitment of RPA occurs after pol/prim, a number of investigators (58–60) have found that in the Xenopus oocyte system, as well as in Saccharomyces, loading of pol/prim onto chromatin is dependent on prior binding by RPA. There are several obvious differences between the SV40 and the cellular DNA replication systems that could account for this disparity. First, the viral system depends on T antigen for helicase activity whereas the cellular system relies on the MCM proteins. The first provides for unregulated multiple firing of the origin; the second is regulated and fires only once per cell cycle, and this basic functional difference may require a different loading order. Second, RPA, pol/prim and topo I can bind directly to T antigen in the SV40 system; it is not known if these proteins bind to MCM directly and it is quite possible that they are recruited by binding to any of the other multiple components of the pre-replication complex. Third, although a less likely possible explanation, the SV40 DNA used here is not associated with histones in chromatin unlike cellular chromosomal DNA. Any or all of these explanations could account for this difference.
ACKNOWLEDGEMENTS
This work was supported by grant CA36118 from the National Cancer Institute to D.T.S.
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