Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system

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Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system
  See discussions, stats, and author profiles for this publication at: Identification of a mammalian RNA polymerase I holoenzyme containingcomponents of the DNA repair/replication system Article   in   Nucleic Acids Research · October 1999 DOI: 10.1093/nar/27.18.3720 · Source: PubMed CITATIONS 64 READS 37 5 authors , including: Some of the authors of this publication are also working on these related projects: transcription by RNA polymerase I   View projectProbing a black box: Pol-I transcription in unicellular parasites.   View projectRoss HannanAustralian National University 221   PUBLICATIONS   5,576   CITATIONS   SEE PROFILE Alice CavanaughGeisinger Health System 42   PUBLICATIONS   1,595   CITATIONS   SEE PROFILE William M HempelAtomic Energy and Alternative Energies Commission 52   PUBLICATIONS   1,359   CITATIONS   SEE PROFILE Lawrence I RothblumUniversity of Oklahoma Health Sciences Center 133   PUBLICATIONS   6,081   CITATIONS   SEE PROFILE All content following this page was uploaded by Ross Hannan on 28 May 2014.  The user has requested enhancement of the downloaded file.   3720–3727   Nucleic Acids Research, 1999, Vol. 27, No. 18 © 1999 Oxford University Press Identification of a mammalian RNA polymerase Iholoenzyme containing components of the DNArepair/replication system Ross D. Hannan, Alice Cavanaugh 1 , William M. Hempel, Tom Moss andLawrence Rothblum 1, * Cancer Research Centre and Department of Biochemistry, Laval University, Hotel-Dieu de Quebec, 11 Cote du Palais,Quebec G1R 2J6, Canada and  1 Henry Hood Research Program, Penn State College of Medicine, Weis Center forResearch, Danville, PA 17822-2618, USA Received May 21, 1999; Revised and Accepted July 21, 1999 ABSTRACTTraditionalmodelsfortranscriptioninitiationbyRNApolymerase I include a stepwise assembly of basictranscription factors/regulatory proteins on the corepromotertoformapreinitiationcomplex. Incontrast,we have identified a preassembled RNA polymerase I(RPI) complex that contains all the factors necessaryand sufficient to initiate transcription from the rDNApromoter in vitro  .ThepurifiedRPIholoenzymecontainsthe RPI homolog of TFIID, SL-1 and the rDNA tran-scription terminator factor (TTF-1), but lacks UBF, anactivator of rDNA transcription. Certain componentsoftheDNArepair/replicationsystem,includingKu70/80,DNA topoisomerase I and PCNA, are also associatedwith the RPI complex. We have found that the holo-enzyme supported specific transcription and thatspecific transcriptionwasstimulatedby theRPItran-scription activator UBF. These results support thehypothesis that a fraction of the RPI exists as a pre-assembled, transcriptionally competent complexthat is readily recruited to the rDNA promoter, i.e. asa holoenzyme, and provide important new insightsinto the mechanisms governing initiation by RPI.INTRODUCTION RNA polymerase can be purified from  Escherichia coli  in twoforms. Only one of these protein complexes, referred to as theholoenzyme, is capable of initiating transcription from genepromoters. Recent studies have demonstrated that eukaryoticRNA polymerase II (RPII) can be isolated in high molecularweight complexes (2000 kDa) associated with various com-ponents of the transcription machinery as well as DNA repairenzymes (1–7). Some preparations contain both RPII and components of the general transcription machinery, i.e. TBP(TFIID), required for transcription initiation (3,7); this form of  RPII would represent a holoenzyme. On the other hand, somepreparations contain components of the general transcriptionapparatus, but are devoid of TBP and are incapable of specificinitiation unless supplemented with TBP (1,5). It has been sug- gested that the holoenzyme preparations might result from theco-purification of an RPII complex and a general transcriptionfactor complex, because the ligand used in the purification, inthis case CDK7 (3), is a component of both complexes (6). RNA polymerase III can also be isolated as a holoenzymewhich is capable of specific transcription initiation (37).Recent reports have demonstrated the existence of variousforms of RNA polymerase I (38–40). However, there are some significant differences in the composition of the various prep-arations which may reflect differences in the mode of isolationor the species from which they were purified. For example, themammalian polymerase I complex isolated by Seither  et al. (38) contained the accessory rDNA-specific transcription factorUBF, but was not self-sufficient for promoter-dependent tran-scription. In contrast, the  Xenopus laevis  RNA polymerase Icomplex purified by Albert  et al.  (39) did not contain detectableamounts of UBF, but was able to autonomously initiate tran-scription  in vitro .We have previously reported an effective scheme to affinitypurify RNA polymerase I from nuclear extracts of an N1S1cell line that expresses FLAG-tagged A127, the second-largestsubunit of rat RPI (8). In the present study, in an attempt toclarifythe abovediscrepancies,wehaveutilized acombinationof both FLAG-affinity purification and gel filtration to examinethe possibility that the transcription factors required for specificinitiation by RNA polymerase I can be purified as componentsof a complex that includes the polymerase itself. Both westernanalysis of the complex and the demonstration that the com-plex supported specific transcription confirmed that the factorsrequired for initiation had co-purified with RNA polymerase I.In addition, the isolated complex responded to UBF, an activatorof rDNA transcription (9; reviewed in 10). As this purification scheme relies upon purification of RPI itself, it is unlikely that wehaveco-purifiedcomplexesthatdonotcontainRPIalongwiththe *To whom correspondence should be addressed. Tel: +1 570 271 6662; Fax: +1 570 271 6701; Email: lrothblum@shrsys.hslc.orgPresent addresses:Ross D. Hannan, Baker Medical Research Institute, PO Box 6492, Melbourne, Victoria 8008, AustraliaWilliam M. Hempel, Center d’Immunologie, INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex, France   Nucleic Acids Research, 1999, Vol. 27, No. 18   3721 RPI holoenzyme. Thus, in these experiments we establish thatRNA polymerase I can be purified as a true holoenzyme. MATERIALS AND METHODS Maintenance of cell lines N1S1C3 cells are a derivative of rat N1S1 cells (rat Novikoff hepatoma cells adapted to tissue culture; ATCC CRL 1604)that express a construct containing the entire coding region of the A127 subunit of rat RPI tagged with the FLAG epitope atthe N-terminus (8). N1S1C3 and wild-type N1S1 cells wereroutinely maintained in spinner flasks containing RPMI 1640with 5% horse serum and the appropriate antibiotic asdescribed (8). Preparation of nuclear extracts Nuclear extracts were prepared from cultured N1S1 andN1S1C3 cells and rat Novikoff hepatoma ascites cells as pre-viously described (11). The extracts were frozen in liquid N 2 and stored at –80 ° C. Sephacryl HR500 gel filtration chromatography Nuclear extracts of rat Novikoff hepatoma ascites cells (0.5 ml,11.5 mg/ml) or N1S1C3 cells (0.5 ml, 14 mg/ml) were frac-tionated by chromatography over a Sephacryl HR500 gelfiltration column (1.6  ×  22 cm) equilibrated in solution D(20 mM HEPES, 0.1 M KCl, 0.2 mM EDTA, 20% glycerol,0.5 mM PMSF, 0.5 mM DTT) containing 2.5  µ M distamycin.Aliquots of each 0.5 ml fraction were assayed for either RPIactivity or used in western blots as described below. In somecases fractions were assayed for specific transcription orimmunoprecipitated with anti-FLAG affinity resin and westernblotted as described below. Ligand and immunoaffinity purification of RNApolymerase I RPI complexes containing FLAG-tagged A127 were immuno-purified using 0.2 ml of packed anti-FLAG resin (2.5 mg/ml;ICI/Kodak)equilibratedin C20 buffer(11) containing 200 mMNaCl (C20/200) and 0.1% NP-40. The resin was tumbled for2 h with 5 ml of nuclear extracts, packed into columns, drainedand washed with 100 column vol of C-20/200 containing 0.1%NP-40. Bound proteins were then eluted with C-20/200 con-taining 0.5 mg/ml FLAG peptide. The eluted fractions as wellas the flow-through were analyzed by immunoblot analysis asdescribed below. In some cases the bound proteins were sub- jected to washes in the presence of increasing amounts of NaCl(i.e. 200, 400, 600 and800 mM)prior to elution with the FLAGpeptide.Tocontrolforproteinsthatmightnon-specificallybindtothe FLAG-affinity resin, nuclear extracts from non-transfectedcontrol N1S1 cells were subjected to affinity purification andwestern analysis in parallel with extracts containing taggedproteins.RPI complexes containing FLAG-tagged A127 were immuno-purified from HR500 gel filtration column fractions using0.2 ml of packed anti-FLAG resin. The resin was equilibratedin C-20/200 containing 0.1% NP-40 and then tumbled for 2 hwith pooled HR500 gel filtration column fractions containingeither Holo-RPI (fractions 33–36, see Fig. 3A) or the Core-RPI(fractions 58–63, see Fig. 3A), after which they were packedinto columns and washed with 10 column vol of C-20/200.Bound proteins were then eluted with FLAG peptide andaliquots of the eluted fractions were analyzed by immunoblotanalysis as described. Immunoblotting Protein determinations used the Bio-Rad Bradford assay kitwith bovine serum albumin (BSA) as the standard. Westernblots were carried out as described previously (8). Proteins of interest were detected by incubatingfilters with the appropriateprimary antibodies at dilutions between 1:100 and 1:5000,followed by horseradish peroxidase-conjugated anti-rabbit oranti-mouse antibodies (Amersham). Immunoreactive proteinswere visualized by the enhanced chemiluminescence method(Amersham). The molecular size of the immunodetectedproteins was verified by comparison to the migration of prestained protein markers (Bio-Rad) electrophoresed in parallel.  In vitro  transcription Specific transcription by RPI was carried out essentially asdescribed (11) using nuclear extracts from N1S1 cells, ratNovikoff hepatoma ascites cells and FLAG-affinity purifiedRPI fractions. Specific transcription was carried out in thepresence of 200  µ g/ml  α -amanatin with 10  µ l of the extracts/ fractions and 0.1  µ g of   Eco RI-linearized template DNA,pU5.1E/X(11),whichcontainstherat45SrDNApromoter(–286to+630).The invitro synthesizedRNAwas purifiedandanalyzedby urea–PAGE and autoradiography .  In some cases partiallypurified UBF (HS600), prepared as described (11), wasincluded in the transcription reactions. TFIC was assayed asdescribed (28).The ability of nuclear extracts, immunopurified FLAG-tagged RPI or high molecular weight RPI complexes fractionatedover Sephacryl HR500 to carry out non-specific RNA synthesiswas assayed in the presence of 200  µ g/ml  α -amanatin as pre-viously described (8). Non-specific transcription reactionscontained 25  µ l of the starting extracts or anti-FLAG affinitycolumn eluate and nicked calf thymus DNA as template asdescribed previously (8). The radioactivity incorporated wasdetermined by liquid scintillation spectrophotometry. RESULTS AND DISCUSSION Affinity purification of RPI using FLAG-affinitychromatography The goal of these experiments was to determine whethertranscription factors required for specific initiation by RPI canbe purified as components of a complex that includes thepolymerase itself. RPI and putative RPI-associated complexeswere affinity purified from nuclear extracts of a rat N1S1 cellline (N1S1C3) that expresses a construct containing the entirecoding region of the A127 subunit of RPI tagged with theFLAG epitope at the N-terminus (8). Nuclear extracts derivedfrom either N1S1C3 cells or control, untransfected N1S1 cellswere applied to pre-equilibrated anti-FLAG affinity columnsandafter extensive washing in buffer containing 200 mM NaCland 0.1% NP-40 the columns were eluted with FLAG peptide(Fig. 1A and Materials and Methods). The eluted fractions pre-pared in this manner were subsequently analyzed for RPIactivity and by western blot analysis. To purify ‘core’ RPI   3722  Nucleic Acids Research, 1999, Vol. 27, No. 18  devoid of associated complexes, the affinity columns werewashed with buffer containing 800 mM NaCl prior to FLAGelution (Fig. 1A and discussed below).Prior to carrying out those experiments, it was necessary toeliminate the possibility that such a complex might arise fromthe co-association of the components with DNA fragmentsgenerated during preparation of the extracts. To address thisquestion we fractionated the extracts in the presence of dista-mycin. Distamycin binds in the minor groove of DNA, inhibitsDNA binding and transcription by RNA polymerase II (3) andat 2.5  µ M distamycin inhibits transcription by RPI (Fig. 1B).Subsequently, all affinity purifications were performed in thepresence of 2.5  µ M distamycin.As shown in Figure 2A, eluates from the N1S1C3 columnfractionated in the presence of distamycin contained significantRPI activity. Western blot analysis demonstrated that the RPIactivity co-purified in the same fractions as did the FLAG-taggedA127 subunit and the untagged core subunits of RPI, A194 (8)and AC40 (12) (Fig. 2A). As expected, the anti-FLAG immuno- purified RPI complex also contained significant amounts of PAF53 (Fig. 2B), a polymerase-associated factor thought to beinvolved in the regulation of RPI transcription initiation(8,12,13). In contrast, neither RPI activity nor RPI subunits were detected in eluates from control N1S1 cells. The affinity-purified RPI complex contains bothtranscription initiation and transcription terminationfactors We next examined what other components of the RPI tran-scription apparatus might co-purify with the affinity-purifiedRPI. In addition to RPI, a  trans -acting factor, selectivity factor-1(SL-1), is absolutely required for initiation of transcriptionfrom the rDNA promoter (14,15). SL-1 consists of TBP andthree TATA-associated factors (TAFs) specific for rDNA tran-scription (16) and is the RPI homolog of TFIID. Western blotanalysis oftheanti-FLAGimmunopurifiedRPIcomplex,isolatedin the presence of 2.5 µ M distamycin, demonstrated the presenceof TBP, TAF I 110 and TAF I 48, three subunits of SL-1 (16),indicating that this factor had co-purified with the RPI complex(Fig. 2B).Termination of mammalian rDNA transcription is effectedby the binding of a nucleolar protein, TTF-1 (130 kDa), to arepetitive sequence motif (the Sal box) within the intergenicspacer (17). Sal box-mediated termination results from a specificinteraction between TTF-1 and RP1 (18). As the two proteinscan interact, we examined the possibility that TTF-1 might beassociated with the affinity-purified RPI. As shown in Figure 2C,western blot analysis of the anti-FLAG immunopurified RPIcomplex isolated in the presence of 2.5 µ M distamycin demon-strated the presence of TTF-1, indicating that this factor was acomponent of the affinity-purified RPI complex.A second  trans -acting factor, upstream binding factor(UBF), is required for efficient transcription from the 45SrDNA promoter (10,14,15). Current evidence suggests that UBF functions as an activator of rDNA transcription (9).  Invitro  assays for protein–protein interactions have demonstratedthat UBF has the potential to interact with RPI (13,19) and TAF I 48 (20) and UBF has been found in one RPI holoenzymepreparation (37). Co-immunoprecipitation experiments usinganti-UBF antisera have confirmed the interaction betweenUBF and SL-1 (21), but have failed to detect an interactionbetween UBF and RPI (8,21). In agreement with the latterobservation, western blots demonstrated that UBF did not co-purify with the RPI complex (Fig. 2C). Although the titers of the anti-UBF and anti-A127 antisera used in these experimentswere approximately equal, the anti-UBF antiserum failed todetect UBF in the RPI complexes that clearly contained A127(even after prolonged exposures). These results confirm ourprevious studies (8,21) and suggest that either UBF–RPI and/orUBF–SL-1-RPI complexes do not form in the absence of DNAor they do not survive the relatively gentle immunopurificationprocedures used in these experiments. In addition, it is interestingto note that the RPI complexes did not contain either TFIIF(RAP 30/74) or SRB11 (CDK8), components of the RPIIholoenzyme(1–6;Fig.2C).Theseresultsdemonstratedifferences between the RPI and RPII holoenzymes, as well as the specificityof the immunopurification procedure, i.e. components of theRPII holoenzyme do not co-purify with the RPI complex. B. Figure 1.  ( A ) Schematic of the procedure used to affinity purify RPI (Core-RPI)and RPI-associated complexes (Holo-RPI) from nuclear extracts of NISIC3cells. ( B ) Distamycin inhibits transcription by RNA polymerase I.  In vitro transcription, using the rat rDNA promoter (pU5.1E/X), was carried out asdescribed (11) in the presence of the indicated amounts of distamycin.   Nucleic Acids Research, 1999, Vol. 27, No. 18   3723 A high molecular weight RPI complex can be identified bymolecular sieve chromatography We then determined if this RPI complex could be identified bymolecular sieve column chromatography. Nuclear extractswere fractionated over a Sephacryl 500-HR gel filtration columninthe presence of2.5 µ M distamycin.The majority of theRNApolymeraseIactivityelutedwithanequivalentmolecularmass of  ∼ 500–600kDa,thesizepredictedforcoreRPI(Fig.3A).However, ∼ 5–10% of the RPI activity chromatographed with an apparentmass of 2000 kDa. Western blots of both forms of RPI demon-strated the presence of the 194 kDa subunit (Fig. 3A, anti-A194,lower panel; note that the left and right sides of the panel areexposed for different lengths of time) and other core subunitsof RPI (results not shown), confirming that these fractionscontained RPI. In a complementary series of experiments,nuclear extracts of NISIC3 cells expressing FLAG-taggedA127 were fractionated over a Sephacryl 500-HR gel filtrationcolumn in the presence of 2.5  µ M distamycin. Immunoaffinitypurification of the column fractions using anti-FLAG resindemonstrated that SL-1 was associated with the ‘high’ molecularweight RPI complex (Holo-RPI) but did not co-purify with the‘low’molecularweightcomplex(Core-RPI)(Fig. 3B).Consideredwith the affinity purification experiments presented in Figure 2,these results demonstrate that a subset of RPI (<10%) can beisolated in the form of a high molecular weight complex pre-assembled with some of the basal components of the RPI tran-scription machinery. The purified RPI complexes support specific transcriptionfrom the rDNA promoter and are responsive to activators The presence of the transcription initiation factor SL-1 in theimmunopurified RPI complex and high molecular weight RPIcomplex obtained by molecular sieve column chromatographyledustoexamineifthecomplexeswouldbecapableofaccuratelytranscribing rDNA. Cell-free transcription assays demon-strated that the high molecular weight complex isolated bymolecular sieve column chromatography could support specifictranscription from the rat rDNA promoter in the absence of exogenously added SL-1 (Fig. 4A, fractions 34 and 35). Incontrast, the low molecular weight complex (Core-RPI) isolatedby molecular sieve column chromatography did not supportspecific transcription (results not shown). Holoenzyme purifiedby FLAG immunoaffinity chromatography from extracts of N1S1C3 cells also supported specific transcription (Fig. 4B,lane 1). It is important to note that the holoenzyme retains itsability to respond to UBF, an activator of rDNA transcription(Fig. 4B, lanes 2 and 3; 9). A. B. C. Figure 2.  Analysis of the proteins that assemble with the FLAG-tagged A127 subunit of RPI. RPI complexes were affinity purified from nuclear extracts of a ratN1S1 cell line (N1S1C3) that constitutively expresses a construct containing the A127 subunit of RPI tagged with the FLAG epitope at the N-terminus (8). ( A ) Nuclearextracts derived from either N1S1C3 cells or control N1S1 cells were applied to anti-FLAG affinity columns (Ld, column load, FT, flow-through material). Thecolumns were eluted with FLAG peptide (0.5 mg/ml) in C20/200 buffer (20 mM HEPES, 5 mM MgCl 2 , 0.2 mM EDTA, 5 mM DTT, 100 mM PMSF, 20% glycerol,containing 200 mM KCl) and the eluted fractions (E2–E5) analyzed for both RPI activity (8) and for the presence of different polypeptides by SDS–PAGE andwestern blot analysis. ( B ) Nuclear extracts of N1S1C3 (lane 1) and N1S1 (lane 2) cells were fractionated by affinity chromatography over immobilized anti-FLAGantibody in the presence of 2.5  µ M distamycin as described above and analyzed by western blotting for the subunits of RPI (A194, A127, PAF53 and AC40) or thesubunits of SL-1 (TAF I 110, TAF I 48 and TBP). A sample (5  µ l) of partially purified ( ∼ 300-fold) SL-1 (HS1000) (11) was included in lane 3 as a positive controlfor SL-1 (note that this fraction does not contain detectable RPI activity). CRM, material unrelated to TAF I 110 that cross-reacts with the anti-TAF I 110 antibodies.( C ) Nuclear extracts of N1S1C3 (lane 1) and N1S1 (lane 2) cells were fractionated by affinity chromatography over immobilized anti-FLAG antibody in the presence of 2.5  µ M distamycin as described above and analyzed by western blotting for the presence of TTF, UBF, TFIIF (RAP 30/74) and SRB11 (CDK8). A sample (20  µ g)of a N1S1 cell nuclear extract (N.E) was included in lane 3 as a positive control.
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