Báo cáo Y học: Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme pptx

Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme Linda Holland, Michael Downey, Xiaomin Song*, Laura Gauthier, Patricia Bell-Rogers and Krassimir Yankulov Department of Molecular Biology and Genetics, University of Guelph, Ontario Canada Minichromosome maintenance (MCM) proteins are part of the replication licensing factor (RLF-M), which limits the initiation of DNA replication to once per cell cycle. We have previously reported that higher order complexes of mam- malian pol II and general pol II transcription factors, referred to as pol II holoenzyme, also contain MCM pro- teins. In the present study we have analyzed in detail the interaction between MCM2 and pol II holoenzyme. N- and C- terminal deletions were introduced into epitope-tagged MCM2 and the truncated proteins were transiently expressed in 293 cells. Affinity chromatography was used to purify RNA pol II holoenzyme and histone binding MCM complexes. We found that amino acids 168–230 of MCM2 are required for its binding to pol II holoenzyme in vivo.We also showed that bacterially expressed amino acids 169–212 of MCM2 associate with pol II and several general tran- scription factors in vitro. Point mutations within the 169–212 domain of MCM2 disrupted its interaction with pol II holoenzyme both in vitro and in vivo.Thisregionisdistinct from the previously characterized histone H3 binding domain of MCM2. Keywords: MCM2; RNA polymerase II holoenzyme; his- tone. Large protein complexes, which contain RNA polymerase II as well as the general pol II transcription factors (GTFs) TFII A, B, D, E, F, and H [1] and other proteins have been isolated from yeast, mammalian and amphibian cells [2–8]. TheyarereferredtoaspolIIÔholoenzymeÕ. Some of the components of pol II holoenzyme make direct or indirect contacts with the C-terminal domain (CTD) of the largest subunit of pol II. Antibodies against the CTD disrupt the yeast holoenzyme into core pol II and a mediator subcom- plex, which contains the SRB and MED proteins [7–9]. A similar treatment of pol II holoenzyme from HeLa cells also disrupts its interaction with several of the GTFs [6]. In higher eukaryotes the CTD mediates the interaction with complexes that contain homologues of the yeast SRB and MEDproteinssuchasSMCC[10]orNAT[11].Itis believed that pol II holoenzyme is a functionally significant complex, which is responsible for transactivator-stimulated transcription in vivo. It has been shown that the srb4 and srb6 genes are essential for expression of most mRNAs in budding yeast [12]. Other holoenzyme components such as SRB 2, 5, 7–11, SWI/SNF proteins, SIN4, RGR1, MED2, MED9/CSE2, MED10/NUT2, MED11, GAL11, PGD1 and ROX3 [7,9,13–16] are not essential for transcription of most genes but do contribute to the response to transacti- vators and repressors (reviewed in [17,18]). In addition to its role in the response to transcriptional regulators, pol II holoenzyme may be involved in integrating transcription with RNA processing, DNA repair and replication. In support of this idea, the DNA repair factors DNA pol e, XPC, XPF, XPG, Ku, RAD51 [3], BRCA1 [19]; RNA helicase A [20]; the replication factors RP-A, RP-C [3] and MCM proteins [6]; and the cleavage/polyadenylation factors CPSF and CstF [21] have been identified in mammalian pol II holoenzyme preparations. There are significant differences in the composition of pol II holoen- zymes that have been purified by different procedures indicating that this complex is capable of interacting with a variety of proteins and that there might be multiple forms of pol II holoenzyme in vivo [22]. MCM proteins, previously characterized as components of the replication licensing factor M (RLF-M) [23–26], were also found to associate with pol II holoenzyme in higher eukaryotes [6]. It is believed that RLF-M is acting to limit replication of genomic DNA to a single round per cell cycle [27]. As predicted by the licensing model, most MCMs are released from chromatin during S phase and re-associate at the end of mitosis [28–33]. In addition to promoting initiation of DNA replication, MCMs also seem to function in replication fork movement [28,34,35]. The MCM4,6,7 subcomplex possesses DNA helicase activity [35–38], which has been implicated in both initiation and fork movement. In addition, MCM complexes bind with high affinity to core histone H3/H4 dimers [39,40] and to HBO1 [41], via distinct domains in the N-terminus of MCM2 indicating a possible Correspondence to K. Yankulov, Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Fax: + 1 519 8372075, Tel.: + 1 519 8244120, ext. 6466, E-mail: yankulov@uoguelph.ca Abbreviations: MCM, minichromosome maintenance; CTD, carboxy-terminal domain (of the largest subunit of RNA poly- merase II); GTF, general transcription factor; SMCC, SRB/ MED-containing cofactor; NAT, negative regulator of activated transcription; RLF-M, replication licensing factor M; TBP, TATA box binding protein; HBO1, histone acetyltransferase binding to ORC; ORC, origin recognition complex; FCS, fetal calf serum; GST, glutathione S-transferase; TFIIS, transcription factor II S. *Present address: Pharmacia Corporation, AA215/AA2C, 700 Chesterfield Parkway, Chesterfield, MO 63198, USA. (Received 11 June 2002, revised 26 August 2002, accepted 30 August 2002) Eur. J. Biochem. 269, 5192–5202 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03224.x chromatin remodeling function. MCM2, but not other MCM proteins, also interacts with cdc6, a component of the replication preinitiation complex [42]. The significance of these protein interactions and the precise biochemical role of MCMs in regulating DNA replication remain unclear. There are certain indications that MCMs might be involved in pol II transcription in higher eukaryotes. We have shown that antibodies against MCM2 inhibit pol II, but not pol III, transcription from injected template plasmids in Xenopus oocytes [6]. Two other studies have demonstrated that an interaction between MCM5 and the activation domain of Stat1a is essential for the expression of IFN-c responsive genes [43,44]. On the other hand, recruitment of pol II holoenzyme to origins of replication via GAL11 or TBP significantly stimulates replication of minichromosomes in S. cerevisiae [45], suggesting a possible role of pol II holoenzyme in DNA replication. In this paper we have analyzed the interaction between human MCM2 (also called BM28) and pol II holoenzyme. We report that MCM2 binds to pol II holoenzyme via a sequence in its N-terminal domain. This region is positioned between the site of interaction with histone H3 and the putative HBO1 binding site. MATERIALS AND METHODS Plasmids All plasmids for expression of recombinant human MCM2 encode N-terminally FLAG-tagged polypeptides. pFLAG-MCM2(FL) contains the EcoRI fragment of pBSBM28 ([46], EMBL accession no. P49736), cloned into the EcoRI site of pFLAG-CMV-2 (Sigma). This plasmid and all its derivatives encode MDYKDDDDK LAAANSAESSESFT followed by different MCM2 frag- ments. pFLAG-MCM2(1–197), pFLAG-MCM2(1–247), and pFLAG-MCM2(1–511) were generated by deleting the Sal I, the Bgl II, and the EcoRV fragments from pFLAG-MCM2(FL), respectively. pFLAG-MCM2(1– 167) was generated by subcloning the EcoR1-Dra III fragment of pBSBM28 into EcoRI-Sma I linearized pFLAG-CMV-2. pFLAG-MCM2(1–230) was generated by deleting the BsaAI-Sma I fragment from pFLAG- MCM2(1–247). pFLAG-MCM2(198–892) contains the Sal I fragment of pBSBM28 [46] cloned into the Sal I site of pFLAG-CMV-2 and encodes MDYKDDDDK LAAANSSIDLISVPV followed by amino acids 198–892. pFLAG-MCM2(345–892) contains the Fse I-SmaIfrag- ment of pBSBM28 [46] cloned into Not I-Sma I linearized pFLAG-CMV-2 and encodes MDYKDDDDKLA fol- lowed by amino acids 345–892 of MCM2. All expression plasmids were purified by anion exchange (Qiagen) prior to transfection. pGEX-MCM2(169–212) contains the sequence encoding amino acids 169–212 of MCM2 attached in frame to GST. Site-directed mutagenesis of the pGEX-MCM2(169–212) and pFLAG-MCM2(1–230) plasmids was conducted using the Quikchange site-direc- ted mutagenesis kit (Stratagene). 5¢-CCGCTTCAA GAACTTCCCGGGCACTCACGTCAC-3¢ was used as a primer to introduce changes from LR to PG at positions 192/193, and 5¢-GCCACGGCCACAACGAG CTCAAGGAGCGCATCAGC-3¢ was used to introduce changes from VF to EL at positions 203/204. Point mutations were confirmed by nucleotide sequencing. Antibodies Anti-(Pol II CTD) (8WG16) [47], anti-(BM28-N) directed towards the N-terminus of MCM2 [32], anti-TBP [6], anti- TFIIB [6], anti-CPSFp160 [6], and anti-(Xenopus ORC2) [48] were described previously. Anti-MCM was generated against a highly conserved peptide sequence VVCI DEFDKMSDMDRTA, which is shared between all MCM proteins [49]. The antibody was affinity purified on antigenic peptide coupled to Affigel-10 (Bio-Rad). Anti- p62(TFIIH) was raised against full length human p62 expressed in E. coli. The anti-FLAG antibody M2 was purchased from Sigma. The anti-CycC was from Santa Cruz Biotechnology. Expression of recombinant MCM2 proteins in human embryonic kidney fibroblast cells (293) HEK 293 cells were grown in 15 cm plates (Costar) to 50% confluency in DMEM medium supplemented with 10% fetal bovine serum and antibiotics (100 unitsÆmL )1 penicillin and 100 lgÆmL )1 streptomycin). Each plate was transfected with 20 lg of pFLAG-CMV-MCM2 expression plasmid plus 20 lg of carrier plasmid (pBS) using calcium phos- phate-precipitation. Transfection efficiency was between 30% and 50% as monitored by the expression of green fluorescent protein from pEGFP-C2 (Clontech). Preparation of whole-cell extract Cells were harvested 36–48 h after transfection and whole- cell extract was prepared by lysis in hypotonic buffer and 0.41 M (NH 4 ) 2 SO 4 extraction as described previously [6]. Prior to chromatography, each extract was buffer exchanged in a 10DG column (Bio-Rad) to chromato- graphy buffer (CB) (10 m M Hepes 7.9, 0.2 m M EDTA, 0.2 m M EGTA, 5 m M 2-glycerophosphate, 1 m M Na 3 VO 4 , 1m M NaF, 1 m M benzamidine, 1 m M dithiothreitol, 50 l M ZnCl 2 ,1lgofpepstatinÆmL )1 ,1lg of leupeptinÆmL )1 , 2 lg of aprotininÆmL )1 , 12% glycerol, 0.05% NP-40) plus 50 m M NaCl and clarified by centrifugation for 15 min at 21 000 g. Protein concentration of the extracts after dialysis was 5–10 mgÆmL )1 . GST-TFIIS affinity chromatography RNA polymerase II holoenzyme was purified by GST- TFIIS affinity chromatography as described previously [6]. Briefly, Glutathione S-transferase (GST), and GST-TFIIS (residues 1–301 of mouse transcription factor IIS) were expressed in E. coli BL21(LysS) and immobilized on Glutathione Sepharose 4B (Pharmacia) at 10 mgÆmL )1 . Mini-columns containing 100 lL of beads were prepared. Extracts from four tissue culture plates were passed through a GST column followed by a GST-TFIIS column. Each column was washed twice with 1 mL of CB plus 50 m M NaCl, then five times with 100 lLofCBplus50m M NaCl, eluted four times with 100 lLCBplus0.325 M NaCl and then four times with 100 lL with CB plus 1 M NaCl. Final 100 lL wash and eluate fractions were precipitated in Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5193 0.8 mgÆmL )1 deoxycholic acid and 20% trichloroacetic acid and then re-suspended in SDS/PAGE sample loading buffer. Histone H3/H4 affinity chromatography H3/H4 histones were purified from HeLa cell nuclear pellets following the protocol of Simon and Felsenfeld [50] and coupled to Affigel-10 (Bio-Rad) at 5 mgÆmL )1 of resin. Bovine serum albumin (Fraction V, Sigma) was coupled to Affigel-10 at a concentration of 5 mgÆmL )1 . Purification of MCM proteins on H3/H4-Affigel beads was carried out as described previously [39] with some modifications. Briefly, flow-through fractions from the GST-TFIIS columns (3 mL at 5 mgÆmL )1 protein) were loaded sequentially to a BSA-Affigel column (100 lL) followed by a histone H3/H4 column (100 lL) equilibrated with buffer A (20 m M Tris/HCl pH 7.5, 0.5 m M EDTA, 1 m M dithiothreitol, 0.1 m M phenylmethanesulfonyl fluoride, and 10% glycerol) containing 0.1 M NaCl. The columns were washed two timeswith1 mLandfivetimeswith200 lL of buffer A plus 0.1 M NaCl, and were eluted with 0.5 M ,0.75 M and 2 M NaCl in buffer A (1 mL, 600 lL, and 2 mL, respectively). Wash and eluate fractions were precipitated in 0.8 mgÆmL )1 deoxycholic acid/20% trichloroacetic acid, then resuspen- ded in SDS/PAGE sample loading buffer. GST-MCM2(169–212) affinity chromatography GST, GST-TFIIS, GST-MCM2(169–212)wt, GST- MCM2(169–212)L192P/R193G, and GST-MCM2(169– 212)V203E/F204L proteins were expressed in BL21(LysS)DE3 cells and coupled to glutathione Sepharose 4B (Pharmacia) at 10 mgÆmL )1 . Each column (250 lL) was loaded with HeLa whole cell extract (10 mgÆmL )1 [6]), columns were extensively washed and eluted with 1 M NaCl. Samples were precipitated with 0.8 mgÆmL )1 deoxycholic acid and 20% trichloroacetic acid and analyzed by Western blotting. Western blotting Proteins were transferred to Immobilon-P membrane (Mil- lipore) by semidry electroblotting. Blots were developed by BM Chemiluminescence Blotting Substrate (Roche) or ECL Plus (Amersham) with horseradish peroxidase coupled to secondary antibody (Sigma or Amersham). For quantita- tion, blots were exposed on an Image Station (Kodak, 440CF) and images were analyzed by Kodak 1D Image Analysis Software. Proteomics tools Multiple sequence analysis was performed by BLAST . Three dimensional structure prediction was carried out by 3 D - PSST (http://www.bmm.icnet.uk/) and Swiss-Model (http://www.expasy.ch/swissmod/SWISS-MODEL.html). Prediction of sites of phosphorylation was by NETPHOS 2.0 (http://www.cbs.dtu.dk/services/NetPhos). Hydrophobicity and charge analysis was performed by PROTPARAM (http://www.expasy.ch/tools/protparam.html). Secondary structure prediction was by JPRED 2 (http://jura.ebi.ac. uk:8888). RESULTS Experimental Strategy Previous experiments have shown that antibodies against MCM2 specifically inhibit pol II transcription in Xenopus oocytes [6]. We decided to search for domain(s) in this polypeptide that might be responsible for the interaction between MCM proteins and pol II holoenzyme [6]. FLAG- tagged human MCM2 deletion mutants (Fig. 1) were expressed in 293 cells and assayed for their ability to copurify with pol II holoenzyme or to bind to histones H3/H4. This approach circumvented problems with bac- terial expression of MCM2 that we had encountered in the past (data not shown). Extracts were prepared from transfected cells and pol II holoenzyme was purified by affinity chromatography using GST-TFIIS as a ligand [5,6]. We had previously shown that about 2% of the total endogenous MCM2 in HeLa cell extract copurified with pol II holoenzyme on GST-TFIIS columns. The flow- through fractions of the GST-TFIIS chromatography, which contained the majority of MCM proteins, were subsequently chromatographed on histone H3/H4-agarose as described [38–40]. The binding of different MCM2 deletion mutants to GST-TFIIS-Sepharose or H3/H4 histones relative to endogenous MCM2 and other MCM proteins was analyzed by Western Blotting. Plasmids encoding for recombinant MCM2 polypeptides (Fig. 1) were transfected into 293 cells and expression was Fig. 1. Scheme of constructs for the expression of FLAG-tagged MCM2 deletion mutants. Human MCM2 DNA sequences encoding the indicated amino acid residues in the full length protein were cloned into pFLAG-CMV-2. All sequences contain a N-terminal FLAG tag. (+) denotes that binding of the expressed protein to pol II holoenzyme or histone H3/H4 was observed. (–) denotes that no binding was observed. (–/+) denotes very weak binding. 5194 L. Holland et al. (Eur. J. Biochem. 269) Ó FEBS 2002 allowed to proceed for 36–48 h. Whole cell extracts were prepared as described previously [6]. Under these conditions all recombinant MCM2 polypeptides were expressed and extracted at levels, which were roughly equal to the endogenous MCM2 with the exception of MCM2(198– 892), which was expressed at somewhat lower levels (Fig. 2 and data not shown). Binding of MCM deletion mutants to GST-TFIIS beads GST-TFIIS affinity chromatography was used to purify RNA pol II holoenzyme and associated MCM proteins from whole cell extracts. Each extract was passed through a control column containing GST alone and then loaded on a GST-TFIIS column. Both columns were washed extensively with low-salt buffer and eluted with 0.325 M NaCl and then with 1 M NaCl. Western blots of the load, flow-through, wash, 0.325 M eluate, and 1 M eluate fractions are shown in Fig. 3. As reported previously [5,6], pol II bound to GST- TFIIS and eluted as two distinct fractions at 0.325 M NaCl, which corresponds to pol II holoenzyme, and 1 M NaCl, which corresponds to core pol II (Fig. 3, lanes 1–9). The amounts of pol II in the 0.325 M NaCl eluates were similar between different extracts as detected by an antibody against its largest subunit [5,6]. There were noticeable differences in the intensity of pol II signal in the 1 M eluate probably because of the limiting quantities of antigen in this fraction that occasionally might be below the sensitivity of our antibody. Importantly, in the 0.325 M NaCl eluate from all extracts we detected comparable amounts of endogenous Fig. 2. Expression of FLAG-tagged MCM2 deletion mutants in 293 cells. Plasmids encoding N-terminally FLAG-tagged MCM2 deletion mutants were individually transfected in 293 cells and expressed for 36–48 h. Whole cell extracts were prepared as described in Materials and methods and 90 lg were loaded per lane. Proteins were separated on 10% SDS/PAGE gels and analyzed by Western blotting with anti- FLAG Ig. Fig. 3. Analysis of binding of FLAG-tagged MCM2 deletion mutants to GST-TFIIS. Affinity columns (100 lL) containing GST (10 mgÆmL )1 )or GST-TFIIS (10 mgÆmL )1 ) were loaded in series with 20–40 mg of 293 whole-cell extract, washed extensively and eluted with 0.325 M NaCl and then with 1 M NaCl. 0.33% of the Load (L), flowthrough (FT), and 33% of the final wash (W) and eluate (E) fractions were analyzed by Western blotting with the indicated antibodies. The figure shows one of three independent experiments (lines a–d) or one of two independent experiments (lines e–i). Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5195 MCM2 (Fig. 3, lanes 10–18). Binding of full length FLAG- tagged MCM2 to GST-TFIIS is shown in Fig. 3, row (a), lanes 19–27. Western blotting with anti-FLAG Ig was used to compare the binding of MCM2 deletion mutants to GST- TFIIS relative to the endogenous MCM2 (Fig. 3, lanes 10– 18) and to the full length FLAG-tagged MCM2. Binding to GST-TFIIS was considered positive when signals in the 0.325 M NaCl eluates (Fig. 3, lanes 16 and 26) were stronger than the signals in the final wash fractions (Fig. 3, lanes 15 and 25). While N-terminal deletions such as MCM2(198– 892) and MCM2(345–892) (Fig. 3, rows g, h) displayed deficient association with GST-TFIIS, the C-terminal deletion mutants MCM2(1–230), MCM2(1–247), and MCM2(1–511)(Fig. 3, rows d–f) all coeluted with pol II holoenzyme at comparable levels to that of full length- MCM2(1–892) (Fig. 3, row a). However, further C-terminal deletions MCM2(1–197) and MCM2(1–167) caused an incremental decrease and disappearance of the FLAG signal in the 0.325 M eluate (Fig. 3, rows b, c), while endogenous MCM2 signal in this fraction was similar for all extracts. These initial results suggested that amino acids 168–230 of MCM2 could be involved in the interaction between MCM proteins and pol II holoenzyme. Binding of MCM deletion mutants to H3/H4 dimers MCM proteins bind to histone H3/H4 dimers in vitro via an interaction mediated by the N terminus of MCM2 [38–40]. It is possible that the interaction between MCMs and the pol II holoenzyme is mediated by histones, which could be recruited to the holoenzyme in a specific or nonspecific manner. We tested this possibility by analyzing the binding of the MCM2 mutants to histone H3/H4. The flow-through fractions of the GST-TFIIS columns were loaded sequen- tially on BSA-agarose and H3/H4-agarose columns. The resins were washed with low salt buffer and eluted with 0.5, 0.75, and 2 M NaCl. Load, flow-through, final wash and eluate fractions were analyzed by Western blotting. First we examined the binding of MCMs to the H3/H4 resin using an antibody against the conserved ATP binding domain of all MCM proteins [49]. This antibody cross- reacts with many bands in crude extracts; however, in purified fractions it detects three to five bands, which correspond to MCM proteins [36,39,51]. In the wash fraction and the eluates of the H3/H4 columns we detected three to five bands with the expected mobility of MCM2, MCM3, MCM4, MCM5 and MCM6 (Fig. 4, lanes 5–7). These signals were significantly higher than the correspond- ing background signals from the control BSA-agarose columns (Fig. 4, lanes 1–4). We also detected recombinant MCM2(198–892) and MCM2(345–892), which contained intact ATP binding domain (data not shown). Because this antibody has a low affinity, we did not observe exactly the same profile of bands in all eluates possibly because some of the MCM proteins were present below the threshold of detection. We did not see any MCMs in the 2 M NaCl eluate by this or other anti-MCM Ig (not shown). This observation is in disagreement with the previously reported histone H3/ H4 chromatography experiments, in which the majority of Fig. 4. Analysis of binding of FLAG-tagged MCM2 deletion mutants to H3/H4-agarose. Affinity columns (100 lL) containing bovine serum albumin (BSA, 5 mgÆmL )1 ) and histone H3/H4 (5 mgÆmL )1 ) were loaded in series with 15–30 mg of GST-TFIIS column flow- through, washed and eluted with buffer A containing 0.5 M NaCl, buffer A containing 0.75 M NaCl, and buffer A containing 2 M NaCl. 0.33% of the Load (L), flowthrough (FT), wash (W) and eluate (E) fractions were analyzed by Western blotting with the indi- cated antibodies. In rows a, e, f, g, and h, we pooled all wash fractions and eluates and loaded 33% of each per lane, respectively. In rows b, c, and d, we show 33% of final washand33%ofthepooled0.5 M and 0.75 M eluate. The figure shows one of three inde- pendent experiments (lines a–d) or one of two independent experiments (lines e–i). 5196 L. Holland et al. (Eur. J. Biochem. 269) Ó FEBS 2002 MCMs were found in the 2 M salt eluate [38–40]. We do not understand this discrepancy. Nonetheless, our histone H3/H4 resin specifically retained MCM proteins as reported in [38–40] and was considered adequate for further analyses. Next we examined the binding of the MCM2 deletion mutants to H3/H4 relative to the endogenous MCM2 and to the full length FLAG-tagged MCM2. Significant amounts of endogenous MCM2 were found in all histone H3/H4 eluates while no MCM2 was detected in the corresponding eluates from the control BSA-agarose col- umn (Fig. 4, lanes 8–14). In agreement with previous reports [38–40], the N-terminal deletion mutants MCM2(198–892) and MCM2(345–892) did not associate with histone H3/H4 (Fig. 4, lanes 15–21, rows g and h). All other recombinant polypeptides closely resembled the elution pattern of the endogenous MCM2 (Fig. 4, lanes 15–21). Importantly, the MCM2(1–167) and MCM2(1– 197), which did not bind to GST-TFIIS, bound strongly to histones (Fig. 4, lanes 15–21, rows b and c). This second set of experiments clearly demonstrated that the sequence of MCM2, which confers association with pol II holoenzyme (amino acids 168–230, Fig. 3) is distinct from the sequence, which is required for its association with histone H3/H4 (amino acids 1–167, Fig. 4) [38–40]. Binding of pol II holoenzyme to GST-MCM2(169–212) We tested the possibility that amino acids 168–230 of MCM2 were required for binding to pol II holoenzyme by a different procedure. We expressed this peptide as a GST- fusion protein and used it in affinity chromatography experiments with HeLa cell extract. Because the C-terminus of 168–230 contains the peptide VNYEDLA, which is part of the reported HBO1 binding site [41], we decided to further truncate the C-terminus of this sequence to produce GST-MCM2(169–212) fusion protein. The protein was coupled to glutathione beads and HeLa cell extract was passed in parallel through GST, GST-TFIIS and GST- MCM2(169–212) beads, the beads were washed extensively withCBbufferandelutedwith1 M NaCl. The load, flowthrough, final wash and eluate fractions were analyzed by Western blotting (Fig. 5). In the eluates of both GST- TFIIS and GST-MCM2(169–212) columns we detected the largest subunit of pol II together with subunits of the general transcription factors TFIID (TBP), TFIIH(p62) and TFIIB. Other components of pol II holoenzyme such as CycC(SRB10) and CPSF(p160) were present at signifi- cantly lower levels in the GST-MCM2(169–212) eluates relative to the GST-TFIIS eluates. A component of the origin recognition complex (ORC2), which associates with MCM proteins at origins of replication [52], was not detected in the eluates of both columns indicating that the observed signals are not a consequence of contamination by extract or chromatin. In all Western blots very little or no signal from all antigens was observed in the final wash fractions and control GST eluates. In summary, we observed that MCM2(169–212) was binding pol II, TFIID, TFIIH and TFIIB with similar (albeit lesser) efficiency as compared to a bona fide holoenzyme binding ligand, TFIIS [5,6]. This data is consistent with the idea that amino acids 169–212 of MCM2 are binding to some component(s) of RNA polymerase II holoenzyme. Point mutations in the MCM2(169–212) domain disrupt the binding of pol II holoenzyme in vitro and in vivo If the MCM2(169–212) domain is required for binding to pol II holoenzyme, then mutations in this region should disrupt the interactions, which were described in Figs 3 and 5. To test this hypothesis, we substituted conserved hydro- philic and hydrophobic residues in GST-MCM2(169–212) as shown in Fig. 6A. GST, GST-MCM2(169–212)V203E/ F204L, GST-MCM2(169–212)L192P/R193G, and GST- MCM2(169–212)wt were coupled to beads at 10 mgÆmL )1 (data not shown) and assayed for their ability to pull down components of pol II holoenzyme from HeLa extract. Each column was loaded with 100 mg of HeLa whole cell extract, washed extensively with CB, and eluted with 1 M NaCl. Flowthrough, final wash and eluate fractions were analyzed by Western blotting with antibodies against pol II and several general transcription factors (Fig. 6B). Consistent with our previous experiment (Fig. 5), considerable amounts of pol II, TFIID(TBP), TFIIH(p62) and TFIIB were detected in the GST-MCM2(169–212)wt eluate (Fig. 6B, lane 12), while little to no signal was detected in the wash fractions or in the GST control eluate (Fig. 6B, lane 3). GST-MCM2(169–212)L192P/R193G retained sim- ilar or slightly lower amounts of pol II, TBP, TFIIH(p62) and TFIIB (Fig. 6B, lane 9) relative to GST-MCM2(169– 212)wt. A dramatic decrease in the signals from all peptides was observed in the eluate of the GST-MCM2(169– 212)V203E/F204L column (Fig. 6B, lane 6). These results Fig. 5. GST-MCM2(169–212) chromatography. Affinity columns (250 lL) containing GST, GST-MCM2(169–212), or GST-TFIIS (each at 10 mgÆmL )1 )wereloadedinparallelwith50mgofHeLacell extract, washed extensively and eluted with 1 M NaCl. 0.33% of the Load, flow-through, and 40% of the final wash and eluate fractions were analyzed by Western blotting with the indicated antibodies. The figure shows one of two independent experiments. Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5197 clearly indicated that the V203E/F204L substitution dis- rupted the ability of the MCM2(169–212) peptide to bind pol II and general transcription factors in vitro. Next we tested if the same mutations would interfere with the binding of MCM2 to pol II holoenzyme in vivo.We introduced the V203E/F204L and L192P/R193G substitu- tions into the pFLAG-MCM2(1–230) plasmid. FLAG- MCM2(1–230) was the smallest stable deletion mutant, which contained the 169–212 region and retained full capacity to copurify with pol II holoenzyme (Fig. 3). The wild type and mutant proteins were expressed in 293 cells and extracted at comparable levels (Fig. 7A). Each extract was assayed by GST-TFIIS affinity chromatography. Load, flowthrough, wash and 0.325 M NaCl eluate fractions were analyzed by Western blotting with anti-CTD Ig, and anti- MCM2 Ig, which recognized equally well the endogenous MCM2 and all MCM2(1–230) recombinant peptides (Fig. 7A). As observed in our previous experiment (Fig. 3) the largest subunit of pol II and endogenous MCM2 coeluted in the 0.325 M NaCl eluate of the GST-TFIIS column. The amounts of these two proteins in this fraction were similar for all extracts (Fig. 7B, lanes 7 and 14). We then compared the copurification of each MCM2(1–230) mutant relative to endogenous MCM2 and to the MCM2(1–230)wt control (Fig. 7B, row a). MCM2(1– 230)L192P/R193G (Fig. 7B, row c, lane 14) displayed somewhat deficient association with GST-TFIIS, whereas MCM2(1–230)V203E/F203L was almost completely absent from the pol II holoenzyme fraction (Fig. 7B, row b, lane 14). The relative abundance of the wild type and mutant MCM2(1–230) peptides in the eluates from the GST-TFIIS columns was confirmed by Western blot with anti-FLAG antibodies (data not shown). These results are in agreement with the results obtained with GST-fusion proteins in vitro (Fig. 6). We conclude that the V203E/F203L substitution disrupts the association of MCM2 with pol II holoenzyme both in vitro and in vivo. The experiments presented in Figs 6 and 7 further verify the importance of amino acids 169–212 to the interaction between MCM2 and RNA pol II holoenzyme. DISCUSSION MCM2 interacts with pol II holoenzyme We have previously shown that several MCM proteins copurified with pol II holoenzyme preparations from human and Xenopus cells [6]. In this paper we show that a likely site, which mediates this interaction is amino acids 169–212 of MCM2. Our results demonstrate that in vivo expressed epitope-tagged MCM2 deletion mutants bind to GST-TFIIS and elute in the pol II holoenzyme fraction as shown before [5,6] only if they contain amino acids 168–230 (Fig. 3). N- and C-terminal deletions within this region significantly decreased, but did not completely abolish binding to GST-TFIIS (Fig. 3, lines c and g). The C-terminal deletions MCM2(1–167) and MCM2(1– 197) retained their ability to associate with histones H3/ H4 (Fig. 4, lines b and c) as previously reported [38–40]. Therefore, it is unlikely that their deficiency in binding pol II holoenzyme is a result of misfolding or inactivation. We do not have a reliable assay to test the functional status of MCM2(198–892) and MCM2(345–892) (Figs 3 and 4) and other N-terminal deletions beyond amino acid 345 (not shown), which did not bind to GST-TFIIS or histone H3/H4. In a separate set of experiments we show that a GST- MCM2(169–212) ligand binds pol II and several previously characterized components of RNA polymerase II holoen- zyme in vitro with similar efficiency as compared to GST- TFIIS (Fig. 5). These experiments imply that MCM2 peptides might be recruited to the holoenzyme independ- ently of whether they are in a complex with other MCM proteins or not. Furthermore, a previous study [53] indica- tedthattheinteractionbetweenMCM2andMCM4,6,7is located in the C-terminus of MCM2. Previously character- ized complexes of MCM2 such as RLF-M and MCM2,4,6,7 [51,54,55] consist of single molecules of the six or four MCM proteins, respectively. It is therefore unlikely that the deletion mutants were recruited to GST– TFIIS via interactions with complexes that already contain endogenous MCM2. Fig. 6. Point mutations in the MCM2(169–212) domain interfere with the binding of pol II and general transcription factors in vitro. (A) The indicated amino acid substitutions were introduced into the GST- MCM2(169–212) peptide by site-directed mutagenesis. (B) Affinity columns (250 lL) containing GST, GST-MCM2(169–212)V203E/ F204L, GST-MCM2(169–212)L192P/R193G, or GST-MCM2(169– 212)wt (each at 10 mgÆmL )1 ) were loaded with 100 mg of HeLa whole cell extract, washed extensively and eluted with 1 M NaCl. 0.33% of the Flowthrough (FT) and 40% of each final wash (W) and eluate (E) fractions were analyzed by Western blotting with the indicated anti- bodies. The figure shows one of two independent experiments. 5198 L. Holland et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The idea that MCM2(169–212) is a site for interaction with pol II holoenzyme is significantly strengthened by the observation that a double amino acid substitution (V203E/ F204L) within this region disrupted the association of this domain with pol II holoenzyme both in vitro and in vivo (Figs 6 and 7). Taken together, our results strongly suggest that amino acids 169–212, which are not required for the interaction of MCM2 with histones H3/H4 [39,40] (Fig. 4) or HBO1 [41], are involved in the interaction with some component(s) of pol II holoenzyme. We propose that three different sites of MCM2 are involved in independent interactions with histone H3/H4, pol II holoenzyme and HBO1asshowninFig.8. In light of our previous study [6] the most likely explanation of our observations is that MCM2(169–212) associates with some component(s) of pol II holoenzyme directly or via a bridging interaction. The identity of this component is not known. We have shown that antibod- ies against the carboxyterminal domain of the largest subunit of pol II (CTD) disrupt the interaction between pol II holoenzyme and MCM proteins [6]. In addition, MCMs, CPSF, CstF, and TFIIH bound to recombinant CTD [6]. It is possible that the partner of the MCM2(169–212) peptide in pol II holoenzyme is within one of these three factors or it is the CTD itself, however, other components of the pol II holoenzyme should also be considered. Characteristics of the MCM2(169–212) sequence The MCM2(169–212) sequence displays no obvious fea- tures that might suggest possible function (See Fig. 8). It has a pI of 9.18 and evenly distributed positively and negatively charged residues. Jpred2 indicates two stretches of potential helices, however, no homology to previously characterized protein–protein interaction domains and no obvious simi- larity with known 3D structures as determined by SWISS - MODEL and 3 D - PSSM were detected. These analyses give us little hint about the nature of this domain and the possible mechanism of interaction with potential target peptides. NETPHOS 2.0 indicated three high score phosphorylation sites in the putative pol II holoenzyme binding sequence of MCM2 (Fig. 8). It is conceivable that interaction between MCMs and holoenzyme is regulated by phosphorylation at the predicted serine residues, but the significance of these residues is yet to be established. The 169–212 amino acid sequence of human MCM2 has highly homologous counterparts in mouse, Xenopus,and Drosophila (Fig. 8), suggesting that a similar interaction between MCM2 and pol II holoenzyme might be taking place in these organisms. The homology with the cognate MCM2 regions in S. pombe, C. elegans and S. cerevisiae is 56, 54 and 46%, respectively, with several highly conserved hydrophobic residues, which are identical in all species (Fig. 8). The sequence similarities do not provide clues as to Fig. 7. Point mutations in the MCM2(169–212) domain diminish binding of pol II holoenzyme in vivo. (A) The amino acid substitutions, shown in Fig. 6A, were introduced into the pFLAG-MCM2(1–230) plasmid by site directed mutagenesis. N-Terminally flag-tagged full-length MCM2(1– 892) (lanes 1 and 5), MCM2(1–230)V203E/F204L (lanes 2 and 6), MCM2(1–230)L192P/R193G (lanes 3 and 7), and MCM2(1–230)wt (lanes 4 and 8) were expressed in 293 cells and whole cell extracts were prepared and 110 lg were loaded per lane. Proteins were resolved on 10% SDS/PAGE gels and analyzed by Western blotting with anti-FLAG (lanes 1–4) and anti-MCM2 (lanes 5–8) Ig. eMCM2, endogenous MCM2. (B) GST-TFIIS affinity chromatography was conducted as described in Materials and methods. 0.33% of the Load (L), flowthrough (FT), and 50% of the final wash (W) and 0.325 M NaCl eluate (E) fractions were analyzed by Western blotting with antipol II CTD and anti-MCM2 Ig. eMCM2, endogenous MCM2. The relative net intensities of bands in Lane 14 are plotted and the position of each MCM2(1–230) mutant and its relative eMCM2 control are indicated. The figure shows one of three independent experiments. Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5199 whether MCM2 and pol II holoenzyme bind to each other in these organisms. In this context it is important that MCM proteins were not detected in any of the S. cerevisiae pol II holoenzyme or mediator complexes despite extensive bio- chemical analyses [2,8,14,56]. Functional significance of the pol II holoenzyme–MCM interaction The functional importance of the established interaction between MCM proteins and pol II holoenzyme remains enigmatic. MCM proteins are components of the DNA replication licensing machinery [24,35]. Their association with pol II holoenzyme could reflect some function of the latter complex in replication. Several findings are in tune with this idea. Recruitment of pol II holoenzyme to origins of DNA replication in S. cerevisiae significantly stimulates their activity [45]. In addition, transcriptional activators, most of which interact with pol II holoenzyme, stimulateDNAreplicationwhentetheredtoviralor cellular origins [57–62]. We found a genetic interaction between pol II CTD and mcm5 andalsoshowedassoci- ation of pol II with origins of DNA replication in S. cerevisiae (K. Yankulov, D. Kramer & R. Dziak., unpublished observations). Control of mammalian origins of replication is less well understood, yet it has been reported that mammalian pol II holoenzyme complexes contains proteins, which function in DNA repair and replication [3,19]. Furthermore, some mammalian origins of DNA replication have been found within mammalian enhancers [63,64], which presumably interact with pol II holoenzyme. Another possibility is that MCM proteins function in pol II transcription. For example, stimulation of IFN-c responsive genes is significantly decreased by mutations in Stat1a, which also preclude the association of this protein with MCM3/MCM5 complex [43,44]. We showed that antibodies against MCM2 inhibit pol II transcription in injected Xenopus oocytes. Effects on DNA replication were not analyzed because these cells do not replicate DNA [6]. We also found some transcriptional deficiencies in mcm5 mutants in S. cerevisiae (K. Yankulov and D. Leishman, unpublished results). Preliminary experiments also indicate consistent inhibition of the expression of a plasmid-borne reporter gene upon overexpression of MCM2 with muta- tions in the holoenzyme interacting domain (data not shown). It seems that the observed association between MCM proteins and pol II holoenzyme could reflect some addi- tional roles of these two complexes in pol II transcription and DNA replication, respectively. At present, the mech- anism of action that signifies this association is unclear. It is conceivable that histones, pol II holoenzyme, and ORC could come in close proximity via contacts with MCM2 to positively (or negatively) regulate origin function. MCM2 may also have some unknown role in mediating pol II– histone contacts. Clearly, addressing these questions requires an in vivo system where effects on DNA replication, transcription, and the cell cycle can be analyzed. The identification of mutations in MCM2, which preclude its interaction with pol II holoenzyme, is therefore a major step towards such a detailed analysis. MCM2(169–212) peptides or full length MCM2 with mutations within the pol II holoenzyme binding region can be tested for dominant negative effects in stably transfected cells. This approach will provide opportunities for a focused functional analysis of the MCM–pol II holoenzyme interaction. ACKNOWLEDGEMENTS We thank N. Thompson and I. Todorov for gifts of antibodies; I. Todorov for the pBS/BM28 plasmid; R. Lu for help with generation of antibodies; R. Mosser, A. Wildeman, D. Evans, J. Bag, G. Harauz, D. Leishman for valuable suggestions and discussion. This study was supported by a grant from Canadian Institutes for Health Research (CIHR no. 36371) to K. Yankulov. Fig. 8. Characteristics of the MCM2(169–212) peptide. 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