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Prohibitin, a tumor suppressor protein, has been shown to repress E2F-mediated transcription and arrest cell cycle progression. While prohibitin has been proposed to regulate cell cycle progression by repressing transcriptional targets of E2F1, it is not clear whether other mechanisms are also involved in mediating the growth arrest. Here we demonstrate that prohibitin can function as a potent inhibitor of DNA replication by interacting with members of Minichromosome maintenance complex of proteins (MCM2-7). The data presented here indicates that prohibitin can physically interact with MCM2, MCM5 and MCM7 in in vitro GST binding assays as well as in MCF-7 cells as seen by immunoprecipitation-Western blot experiments. The association was cell cycle dependent, and more pronounced 4–8 hours after serum stimulation of quiescent cells. Prohibitin associated more robustly with MCM2 and MCM5 compared to MCM7, suggesting that prohibitin mainly interacts with the regulatory subunits of the MCM complex. Confirming these results, prohibitin was found to co-localize with MCM2, MCM5 and MCM7 in MCF-7 cells, as seen by double immunofluorescence experiments. Further, Prohibitin strongly inhibited DNA replication in an in vitro replication assay. These results strongly suggest that prohibitin effectively represses replication by interacting with the components of mammalian replication machinery and this might contribute to the growth regulatory properties of prohibitin.
Maintenance of chromosome integrity requires accurate replication of the genome during S phase of the cell cycle. In mammalian cells, precise DNA replication is initiated at numerous origins distributed throughout the DNA. Origins are licensed for replication in late mitosis and G1 by loading complexes of the minichromosome maintenance proteins 2-7 (MCM 2-7) forming a “pre-replicative complex” (pre-RC).1,2 Prior to that, origins are first marked by the origin recognition complex (ORC), which is composed of six subunits (ORC1-6)3 and serves as a landing pad to employ the DNA replication machinery.4 Once ORC binding takes place, cdc65 and cdt16 associate and function to recruit the MCM proteins, a group of highly conserved proteins essential for the initiation of DNA synthesis.7–9 MCMs are displaced from replication origins during initiation and provide a helicase activity to unwind DNA ahead of the replication fork. Replication origins fire only once in a single S phase and their ability to license new origins is prevented from late G1 through to mid-mitosis.1, 2,10
A number of proteins have been identified and their role recognized in controlling replication, the precise regulatory mechanism for many still remains unclear.11–14 The retinoblastoma protein, Rb is known to inhibit replication in S phase through attenuation of PCNA function.15 It also associates directly with the pre-RC through MCM716 and perhaps subunits of ORC17 whereas p16INK4a disrupts pre-replication complex assembly by inhibiting MCM protein loading in G1.18 Further, the E2F transcription factor regulates the expression of MCM proteins as well as Cdc6 thereby controlling the initiation of replication.19 Prohibitin, a tumor suppressor protein, functions similar to Rb in that it represses E2F family of transcription factors, thereby facilitating its growth-suppressive activity.20,21 Prohibitin plays a key role in the regulation of cell cycle progression20–25 and has potent anti-proliferative activity.26,27 Prohibitin has also been shown to influence the replicative capacity of yeast and inhibit replication in HeLa cells and normal fibroblasts in vitro.28–34 Therefore, we hypothesized that prohibitin could be involved in the regulation of DNA replication as well, in mammalian cells. Since Rb, the major regulator of E2F activity, can regulate replication by associating with components of the pre-replicative complex,16 we decided to assess whether prohibitin can also function in a similar fashion.
The data presented here shows that prohibitin physically interacts with and co-immunoprecipitates very effectively both MCM2 and MCM5, the two components of the MCM complex that regulates replication. MCM 2-7 complex is believed to constitute of two subsets of proteins, one comprising of MCM4, MCM6 and MCM7 reported to possess DNA helicase activity, and MCM2, MCM3 and MCM5 involved in more regulatory activity.35,36 Double immunofluorescence experiments followed by confocal microscopy revealed that prohibitin co-localized with MCM2 and MCM5 in the nucleus, in distinct foci. The association of MCM7 with prohibitin was relatively weaker. Further, prohibitin could significantly inhibit SV40 large T-antigen mediated replication of the pEPI-1 plasmid in the presence of all the cellular components needed for a functional mammalian DNA replication. Together, the data provides evidence that prohibitin can inhibit mammalian DNA replication, probably contributing to cell cycle arrest.
Based on the fact that prohibitin is anti-proliferative and represses active transcription mediated by E2F1, and since Rb family members could regulate replication, we hypothesized that prohibitin could be playing a role in replication as well. To examine this possibility, we tested the ability of prohibitin to interact with the MCM2, MCM5 and MCM7 in an in vitro GST binding assay. GST-Rb and GST-prohibitin proteins were immobilized on a glutathione-sepharose affinity column. 35S-labeled MCM2, MCM5 and MCM7 were synthesized by in vitro transcription-translation reactions and their ability to bind to GST-Rb and GST-prohibitin beads was assessed using our published protocols.37,38 The proteins associated with the beads were resolved on SDS-PAGE and analyzed by autoradiography. It was found that none of the proteins bound to the control GST beads; in contrast, MCM2 (Figure 1A), MCM5 (Figure 1B) and MCM7 (Figure 1C) bound efficiently to GST-prohibitin in vitro. While the association of prohibitin with MCM5 and MCM7 was robust, the association with MCM2 was relatively weaker. GST-Rb was used as a positive control since it is already established that Rb binds to MCM7;16 all the three MCMs could bind to GST-Rb as well. These results suggest that prohibitin, like Rb, associates with MCM2, MCM5 as well as MCM7 and this interaction is probably direct.
In order to evaluate whether prohibitin interacts with MCMs in mammalian cells, additional experiments were conducted on MCF-7 breast cancer cells. The levels of prohibitin and MCM proteins in MCF-7 whole cell lysates were examined by Western blotting. The cells were rendered quiescent by serum starvation, followed by serum stimulation for 2, 4, 6, 8 and 18 hours to facilitate cell cycle progression; quiescent cells serum stimulated for 2, 4, 6 and 8 hours will be in the G1 phase, while those stimulated for 18 hours will be in S-phase. Western blot analysis with respective antibodies revealed higher levels of prohibitin in lysates from quiescent and early G1 cells, with a decrease in prohibitin levels at 8hr and 18hr after serum stimulation; at the same time, levels of MCM2, MCM5 and MCM7 demonstrated a gradual but marginal increase (Figure 2A) during the progression of the cell cycle.
It was next examined whether the prohibitin-MCM protein interactions occurs in vivo and whether the interaction changes during the cell cycle; an immunoprecipitation-Western blot analysis was performed to address this. Lysates from quiescent cells or those stimulated with serum for 2, 4, 6, 8 or 18 hours were immunoprecipitated with a monoclonal antibody to prohibitin and the presence of MCM2, MCM5 and MCM7 in the immunoprecipitates examined by Western blotting. Immunoprecipitation with a secondary IgG was used as the negative control. As shown in Figure 2B, prohibitin was found to associate with MCM2 and MCM5 at all time points, with maximal association at 4 and 6 hours; the interaction was reduced by 18 hours. Prohibitin bound relatively weakly to MCM7 in this experiment, and the interaction was restricted to lysates from cells serum stimulated for 4, 6 and 8 hours. As in the case of MCM2 and MCM5, the interaction with MCM7 was very weak or absent at 18 hours suggesting that prohibitin could possibly be regulating the replication machinery by binding to the regulatory MCMs during G1 phase. The MCM proteins were not detected in the immunoprecipitation done with the control antibody, indicating that the interaction with prohibitin was specific (Figure 2B). It appears that dissociation of prohibitin from the MCM complex in late G1/S phase could be altering the functional status of the pre-replication complex, allowing the replication process to progress.
The MCM helicase is loaded onto chromatin during G1 phase of the cell at replication origins and remain chromatin bound through the initiation of S-phase.39 Considering that prohibitin can associate robustly with MCM2 and MCM5, and to a certain extent with MCM7, we examined whether prohibitin associated with the chromatin-bound MCM complexes during G1 and early S phase. Towards this purpose, quiescent MCF7 cells were serum stimulated for 2, 4, 6, 8 and 18 hours and chromatin-bound and chromatin unbound protein fractions prepared using published protocols.40 Subsequently, levels of chromatin-tethered MCM2, MCM5, MCM7 and prohibitin were assessed by Western blotting. There was a gradual increase in the levels of chromatin bound MCM2, MCM5 and MCM7 during the progression of the cell cycle; interestingly, prohibitin levels remained fairly constant through the course of the experiment (Figure 3A). An immunoprecipitation-Western blot experiment was conducted using the chromatin bound protein fractions to assess whether prohibitin associated with the chromatin-bound MCM proteins. Prohibitin was immunoprecipitated from the chromatin bound fractions with a mouse anti-prohibitin antibody and the presence of MCM2, MCM5 and MCM7 examined by Western blotting. Interestingly, MCM2 and MCM5 showed a strong association with prohibitin in the chromatin bound fraction; the association was decreased (MCM5) or almost absent (MCM2) at the 18 hour time point (Figure 3B). However, there was no detectable association between prohibitin and MCM7 on the chromatin, suggesting that prohibitin could be modulating the replication process through the regulatory subunits of the MCM protein complex. The minimal interaction of prohibitin with MCM7 observed in unfractionated lysates could be due to its association with chromatin-unbound MCM7 or through other MCMs.
To investigate whether prohibitin co-localizes with the regulatory MCM2 and MCM5 as well as with the catalytic MCM7 proteins, a double immunofluorescence experiment was performed on quiescent MCF7 cells, or those stimulated with serum for 2, 4, 6, 8 and 18 hours to promote cell cycle progression. After serum stimulation, soluble nuclear proteins were extracted by incubating the cells in 0.2% Triton X-100 in PBS for 2min or by rinsing three times in the same buffer prior to fixing the cells with formalin, to ensure the interaction of prohibitin is with chromatin tethered MCM’s.41, 42 The localization of the proteins was assessed by confocal microscopy. Prohibitin is known to be distributed in the mitochondria as well as the nucleus; we had shown that prohibitin in predominantly nuclear in MCF-7 cells and it translocates to the mitochondria upon apoptotic signaling. As shown in Figure 4, prohibitin was predominantly localized in the nucleus (Figure 4, left panels), while MCM2 was ubiquitously distributed in the cell (Figure 4, middle panels). This is in agreement with published reports showing that MCM proteins can be detected in both the cytoplasm and the nucleus.43 Overlay of the images showed a distinct co-localization of prohibitin and MCM2 in the nucleus, with the maximal interaction at 4, 6 and 8 hours of serum stimulation (Figure 4, right panels) and dissipated by 18 hours. The interaction appeared as punctate spots in the nucleus.
Similar results were obtained for MCM5 (Figure 5). Unlike MCM2, MCM5 staining appeared to increase upon serum stimulation, with maximal amounts at 2, 4 and 6 hours after serum stimulation. While MCM5 was also ubiquitously distributed in the cell, it was enriched in the nucleus (Figure 5, middle panels), especially at the later time points of serum stimulation. Prohibitin levels were elevated in earlier phases of serum stimulation (Figure 5, left panels). Overlay of the images demonstrated that prohibitin co-localizes with MCM5 at 2, 4, 6 and 8 hours of serum stimulation; there was hardly any interaction in quiescent cells or cells stimulated for 18 hours, which were in S-phase. These experiments suggest that prohibitin co-localizes with the regulatory MCM proteins, MCM2 and MCM5 in the nucleus of MCF-7 cells.
As in the case of MCM2 and MCM5, MCM7 was also ubiquitously distributed in the cell and its levels were elevated upon serum stimulation, with maximum levels observed at 2, 4 and 6 hours after serum stimulation (Figure 6, middle panels). MCM7 showed minimal co-localization with prohibitin (Figure 6, right panels); a low amount of co-localization was observed at 4 and 6 hours after serum stimulation. Thus it appears that prohibitin preferentially associates with MCM2 and MCM5 in the cell; this interaction is predominant during the mid-G1 phase and disappears in late G1/S. The association with the catalytic MCM7 seems minimal in this assay; the observed association can be indirect, through the association with MCM2 and MCM5. The release of prohibitin from MCM2/5 at 18 hours (G1/S) raises the possibility that prohibitin could be involved in the modulation of DNA replication.
Role of prohibitin in regulating cell proliferation is well established26,27 and prohibitin has been shown to inhibit E2F-mediated transcription to inhibit cell proliferation. In addition, it has been shown to influence the replicative capacity in yeast (Schizosaccharomyces) as well as human diploid fibroblasts,28–34 suggesting that additional mechanisms might be contributing to the cell cycle regulation. Given the observation that prohibitin could physically interact with MCM proteins during the G1 phase of the cell cycle, attempts were made to assess whether prohibitin plays a regulatory role in mammalian DNA replication. An in vitro replication assay was performed to address this question. Towards this purpose, cytosolic (S100) and nuclear protein (S300) extracts were prepared from asynchronous MCF7 cells following the protocol by Gruss.44 Both extracts are needed for the assay since cytosolic fraction contains chain-elongating proteins and the nuclear extract contains the necessary replication machinery.45 pEPI-1 plasmid was used as the template for replication since it carries an active CMV promoter and a scaffold/matrix attachment region (S/MAR) essential for extrachromosomal replication; these features make this plasmid amenable to in vitro replication assay.46–48 Control GST as well as GST-prohibitin were expressed in bacteria and purified on a glutathione-sepharose column using standard protocols. To assess whether prohibitin could affect the replication of the pEPI-1, the plasmid was pre-incubated in a reaction mix containing S300 nuclear extract and GST, GST-Phb or commercially available purified Rb protein along with other reaction components for an hour at 37°C; S100 cytosolic extract containing reaction mix was then added to initiate the replication process, and incubation continued for another hour. The efficiency of replication was low with cellular extracts alone; hence commercially available T antigen was used to prime the initiation of replication. The replication products were run on 0.8% agarose and, analyzed by EtBr staining and autoradiography. We first optimized the amount of cytosolic and nuclear extracts to be used to obtain maximum replication. We found that 32μg of S300 and 45μg of S100 extracts gave maximum replication product over other concentrations tested (Figure 7A, lane 2); these concentrations were used in future assays. It was observed that addition of GST-Phb significantly reduced the replicative capacity of pEPI-1 when compared to either S300/S100 extract alone or unprimed GST protein (Figure 7B). Commercially available Rb was used as a positive control to assess the magnitude of replication inhibition (Figure 7C). As expected, there was a significant reduction in replication by Rb, and this was comparable to that observed with GST-prohibitin. The above result strongly implies that prohibitin can suppress replication in mammalian cells, through association with a subset of MCM proteins.
Replication of mammalian genome is stringently regulated by a complex network of proteins that can respond to a variety of upstream signaling molecules.11,49 The core of this regulatory machinery is a set of replication initiation factors that sequentially assemble into pre-RCs at replication origins leading to the chromatin being licensed for replication in the S phase.50, 51 Though the sequential progress of replication is well studied upon replication initiation, it is not clearly understood as to what proteins regulate replication from happening at the appropriate point in the cell cycle. Several lines of evidence suggest a role for Rb and E2F proteins in the DNA replication process. Rb has been shown to bind to several DNA replication proteins such as DNA polymerase α, MCM7, ORC2, etc.16, 52, 53 Rb and E2F have also been shown to localize to the sites of DNA replication early in S-phase.54, 55 E2F DNA binding sites have been identified in the promoter regions of several genes involved in DNA synthesis and replication such as dihydrofolate reductase (DHFR), DNA polymerase, thymidylate kinase, thymidylate synthase, ORC1 and CDC6.56, 57 Studies report that E2F1 does not direct the ORC binding; it restricts its activity through Rb.17 Several studies have revealed a connection of E2F1 activity and DNA replication by demonstrating that replication components like MCMs and DNA polymerase subunits are targets of E2F transcriptional activity.58 Elegant studies from Eric Knudsen’s lab revealed activation of Rb in S-phase cells can disrupt the chromatin tethering of PCNA.15 Other studies revealed Rb can bind to MCM7 and possible units of ORC.16 Recently, it was discovered that Rb controlled the expression of certain replication factors; when Rb is lost replication factors increase in expression and so does their association with chromatin.59 In agreement with these findings, overexpression of E2F was also able to increase expression of replication factors as well as the association with chromatin in the active state. These findings clearly demonstrate how loss of Rb or aberrant E2F activity can lead to deregulation of the replication machinery. Since E2F and Rb have numerous binding partners it is not clear whether they interact directly with the replication machinery, or if they function by recruiting complexes that alter the chromatin structure.
The current understanding on the control of replication initiation has come from studies in yeast and xenopus.60,61 Recently, plasmid pEPI-1 has been recognized to provide a favorable template to study replication under in vitro conditions using mammalian cell extracts.45 This plasmid replicates as an extrachromosomal replicon in mammalian cells and faithfully segregates during mitosis in many cell generations.47 Development of this plasmid has helped elucidate many molecular mechanisms governing mammalian DNA replication; we used this plasmid in our studies to assess the effects of prohibitin. Our interest in the role of prohibitin in replication stemmed from its similarity to Rb in regulating cell growth20 and its ability to bind to E2Fs and repress transcription.21 Since Rb has been shown to bind to MCM7 and prevent DNA replication,16 it was interesting to assess prohibitin could also regulate replication. Indeed, we found prohibitin associated with MCM proteins, mainly with MCM2 and MCM5, in immunofluorescence and co-immunoprecipitation experiments. The association was strong in G1 phase, between 4–8 hours of serum stimulation and then dissipated at 18 hours. The association of prohibitin with MCM proteins could be observed without over-expressing any component in the cell. Our earlier studies had suggested that prohibitin inhibits cell proliferation by repressing the transcriptional activity of E2F-regulated genes. It appears that inhibiting DNA replication is another mechanism by which prohibitin brings about cell cycle arrest.
Prohibitin had been found to inhibit the initiation of DNA synthesis in human diploid fibroblasts upon microinjection of prohibitin mRNA into G0 cells, which were then stimulated to re-enter the cell cycle.23 Prohibitin appeared to exhibit anti-proliferative activity at the G1/S boundary and did not non-specifically inhibit DNA synthesis.25 Although data from several experimental systems show that prohibitin mRNA and protein vary little during the cell cycle, we observed a variation in the expression of prohibitin protein coherent with cell cycle progression in unfractionated MCF7 lysates prepared using a NP-40 lysis protocol. Similar results have been reported recently by Roskams et al,25 wherein they found elevated levels of prohibitin mRNA and protein in G1 phase, which was reduced in S phase. Interestingly, we found that there was no significant change in the levels of prohibitin in the chromatin bound protein fraction. However, MCM levels gradually increased from 0hr to 18hr of serum stimulation. Stoeber K et al62 demonstrated the presence of the essential replication initiation factors ORC, cdc6 and MCM throughout the human proliferative cell cycle. Our results suggest that prohibitin is bound to the chromatin tethered MCMs in the G1 phase and dissociates during S phase. This result, combined with the observation that prohibitin can inhibit replication in vitro, suggests that the association of prohibitin with MCM’s prevents replication from happening prematurely in the cell cycle. Subsequent dissociation of prohibitin, as a result of cell cycle regulated kinases or other signaling molecules, permits DNA replication. This hypothesis is proposed in the model (figure 8), which illustrates that prohibitin is one of the regulatory factors that control replication to happen at appropriate time of the cell cycle. While cyclin D1/CDK4 kinase and not cyclin E/CDK2 or cyclin A/CDK2 specifically triggers the dissociation of Rb-MCM7 interactions,40 cdks did not appear to have a similar effect on serum-mediated inactivation of prohibitin.21 Thus it is not clear whether the dissociation of prohibitin from the MCM proteins is mediated by cyclin dependent kinases or not. Though the role of prohibitin in DNA replication is not completely understood, it appears to be an additional mechanism by which prohibitin regulates cell cycle progression.
MCF7 cells were cultured in DMEM (Mediatech Cellgro) containing 10% FBS. The studies were done on cells that were rendered quiescent by serum starvation for 72hr. Thereafter cells were stimulated with DMEM containing 10% FBS for 2, 4, 6, 8 and 18hr and subjected to various methods of analysis.
MCF7 cells were plated onto poly-D-lysine (Sigma)-coated eight-well glass chamber slides (10,000 cells per well) for immunostaining. Twenty-four to thirty-six hours after plating, the cells were rendered quiescent by serum starving for 72hr followed by releasing with complete DMEM for different times- 2, 4, 6, 8 and 18hr; subsequently, cells were washed with phosphate-buffered saline (PBS) and rinsed with PBS containing 0.2% Triton X-100. Cells were fixed in 10% buffered-formalin for 25min and double immunofluorescence was performed as per the protocol described in Rastogi et al.38 Primary antibodies used were monoclonal prohibitin (NeoMarkers, Inc. Freemont) at 1:200 dilution; polyclonal MCM2 (Aviva Systems Biology), polyclonal MCM5 (Abcam) and polyclonal MCM7 (Santa Cruz Biotech) at 1:100 dilution. Secondary antibodies were goat anti-mouse–Alexa Fluor-488 and goat anti-rabbit IgG–Alexa Fluor-546 or -555 (Molecular Probes). Nuclei were detected using Vectashield mounting medium with DAPI (Vector Laboratories, Inc.). Control experiments demonstrated no cross-reactivity between anti-mouse secondary and anti-rabbit primary antibodies and vice versa; nor was there any detectable staining by secondary antibodies only (data not shown). Cells were visualized with a DM16000 inverted Leica TCS SP5 tandem scanning confocal microscope with a 63x/1.40NA oil immersion objective. 405 diode, 488 Argon and 546 or 555 HeNe laser lines were applied to excite the cells using AOBS line switching to minimize crosstalk between fluorochromes. Images were produced with three cooled photomultiplier detectors and analyzed with the LAS AF software version 1.6.0 build 1016 (Leica Microsystems, Germany).
GST, GST-Rb, GST- prohibitin were purified from bacterial cultures and bound to glutathione-Sepharose beads (Amersham) as described earlier.20 Beads were then washed three times with PBS, and protein integrity was checked by polyacrylamide gel electrophoresis and Coomassie Blue staining. 35S-Methionine-labeled lysates of MCM2, MCM5 and MCM7 were made using the rabbit reticulocyte translation system according to the manufacturer’s directions (Promega). 8μl of labeled lysates was incubated with an equivalent amount of GST or GST- prohibitin or GST-Rb beads in a buffer containing 20mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40, 50mM KCI, 500mM EDTA, and 3mg/ml bovine serum albumin, 1mM dithiothreitol, and 0.5mM phenylmethylsulfonyl fluoride. Samples were incubated for 2hr at 4°C and then washed in binding buffer six times. Bound proteins were eluted in gel loading buffer, resolved by SDS-PAGE gel electrophoresis and bands were visualized by autoradiography. The protein amounts in control input lanes were approximately one-fifth of the total used in the binding assay.
Lysates from MCF7 cells stimulated with serum for different time points were prepared by Nonidet P-40 lysis as described.37 Cells were also subjected to chromatin bound and unbound protein fractionation described below. 100μg of total lysate or 50μg of chromatin unbound and bound proteins were run on 8% SDS-polyacrylamide gel and transferred on nitrocellulose membrane by semidry method to assess the levels of prohibitin, MCM2, MCM5 and MCM7 by Western blotting. Actin (Sigma) was used as loading control for total lysates.
Physical interaction between proteins in vivo was analyzed by IP-Western blotting by using 200μg of total cell lysate or 50μg of chromatin bound fractions for immunoprecipitation with 2μg of the monoclonal prohibitin antibody followed by Western blotting with the following antibodies- polyclonal MCM2, polyclonal MCM5 and monoclonal MCM7 (Abcam). The data presented is a representation of three independent experiments.
Following serum stimulation, cells were harvested and lysed in CSK+ buffer (10mM Pipes (pH7.0), 100mM NaCl, 300mM sucrose, 3mM MgCl2, 0.5% Triton X-100, 1mM ATP, 0.1mM phenylmethylsulfonyl fluoride, 1μg/ml aprotinin, 1μg/ml leupeptin, 1μg/ml pepstatin and 25mM β-glycerol phosphate). Lysates were incubated on ice for 20min and then subjected to low speed centrifugation at 1000×g for 5min. The soluble fractions were removed, centrifuged at 16000×g for 15min at 4°C and collected the supernatant as chromatin unbound fraction. The chromatin pellet from the first low speed spin was washed once with CSK+ buffer containing 1000U/ml DNase I and then digested with the same buffer for 30min at 25°C. The digest was spun at high speed (16000×g) for 15min at 4°C and the supernatant collected as the chromatin bound fraction.40 Soluble and chromatin fractions were then used for direct Western blotting or co-immunoprecipitation.
Soluble proteins were prepared according to Gruss.40,44 Briefly, MCF7 cells from two 100mm dishes were washed twice in cold phosphate-buffered saline, collected in buffer A (20mM Hepes, pH 7.4, 5mM KCl, 1.5mM MgCl2, 0.1mM dithiothreitol) containing 250mM sucrose, and washed twice with buffer A. They were then kept for 10min on ice in a small volume of buffer A and disrupted in a type S Dounce homogenizer. Nuclei were collected by centrifugation at 16,000×g for 10 min. The supernatant was then centrifuged at 100,000×g for 1hr to prepare the S100 extract (6–10μg of protein/μl), which was stored in aliquots at −70°C. The nuclear pellet was washed in buffer A and then kept in buffer A containing 450 mM potassium acetate for 90min on ice. After preclearing (12,000×g for 5min), the supernatant was centrifuged at 300,000×g for 1hr to prepare the S300 extract (2–4 μg of protein/μl), which was stored in aliquots at −70°C.
The assay was performed according to the protocol mentioned in Baltin J. et al.45 As template we used plasmid pEPI-1.46 SV40 large T antigen was purchased from ChimerX, USA (cat # 5800-01). We performed pre-incubations in a total volume of 35μl with S300 nuclear extract (16–64μg) and pEPI-1 DNA (320ng) at 2mM ATP and 80mM potassium acetate in buffer A plus 1μl of Complete, EDTA-free (Roche Applied Science), as protease inhibitor. 3–5μg GST-Phb or GST alone or 0.5μg Rb (QED Biosciences) was also added and incubated at 37°C for 60min. Replication was initiated by the addition of the S100 extract (45–90 μg), 40mM creatine phosphate, 0.6μg/μl of creatine kinase, 30mM potassium acetate, 80μMCTP, GTP, and UTP, 100μM dGTP, dCTP, and dTTP, 30 μM dATP, 1μl 10μCi [α-32P]dATP and 0.5μg T antigen in a total volume of 50μl. Incubation was at 37°C for 60min and then stopped by 30μl of stop mix (60mM EDTA, 2% SDS). DNA was extracted by proteinase K digestion and phenol-chloroform treatment followed by ethanol precipitation. The pellet obtained was re-dissolved in 20μl of milliQ water, run on 0.8% agarose and visualized by EtBr staining. For autoradiography, the gel was dried and exposed to X-ray film. Master mix was prepared to avoid any discrepancies in the addition of any of the components. Creatine phosphate, creatine kinase and potassium acetate were from Sigma. NTPs and dNTPs were from Promega.
This work was supported by the grant CA 77301 from the NCI to Srikumar Chellappan.