MCM proteins are associated with RNA polymerase II holoenzyme

Abstract

MCMs are a family of proteins related to ATP-dependent helicases that bind to origin recognition complexes and are required for initiation of DNA replication. We report that antibodies against MCM2(BM28) specifically inhibited transcription by RNA polymerase II (Pol II) in microinjected Xenopus oocytes. Consistent with this observation, MCM2 and other MCMs copurified with Pol II and general transcription factors (GTFs) in high-molecular-weight holoenzyme complexes isolated from Xenopus oocytes and HeLa cells. Pol II and GTFs also copurified with MCMs isolated by anti-MCM3 immunoaffinity chromatography. MCMs were specifically displaced from the holoenzyme complex by antibody against the C-terminal domain (CTD) of Pol II. In addition, MCMs bound to a CTD affinity column, suggesting that their association with holoenzyme depends in part on this domain of Pol II. These results suggest a new function for MCM proteins as components of the Pol II transcriptional apparatus.

FIG. 1

Anti-MCM2(BM28) antibodies inhibit Pol II transcription in Xenopus oocytes. (A) RNase protection analysis of c-myc and VA1 transcripts from injected oocytes. Anti-BM28-N (0.15 mg/ml; lanes 3 and 4) and anti-BM28-C antibodies (0.22 mg/ml; lanes 5 and 6) were coinjected with mouse c-myc exon I plasmid pSX943 and adenovirus VA1 plasmid pSPVA. Full-length recombinant MCM2(BM28) was coinjected at 0.55 mg/ml (lanes 2, 4, and 6). C, control oocytes injected with 1 mg of BSA/ml. Readthrough (RT) and terminated (TM) transcripts and VA1 Pol III transcripts are indicated. Transcripts from the P1 promoter and those which read around the plasmid protect the full-length probe (P). The RNase protection strategy is diagrammed with P1 and P2 promoters and the T2 terminator. RNase protection signals were quantified from scanned images, and the ratios of myc to VA1 are shown in the histogram, with the control normalized to 100%. (B) RNase protection analysis of pGal5-P2mycCAT and VA1 transcripts. Transcription was activated by injection of recombinant Gal4-AH. Anti-BM28-N (0.15 mg/ml; lanes 3 and 4), anti-Pol II CTD (0.15 mg/ml; lane 5), and anti-RP-A p70 (0.15 mg/ml; lane 6) were coinjected as indicated. Recombinant MCM2(BM28) was coinjected at 0.55 mg/ml (lanes 2 and 4). C, control oocytes injected with 1 mg of BSA/ml. The results were quantified as in panel A. (C) RNase protection analysis of HIV-2 CAT and VA1 transcripts. Anti-ORC1 (1 mg/ml), anti-ORC2 (1.3 mg/ml), anti-Pol II CTD (0.15 mg/ml), anti-TFIIB (0.5 mg/ml), anti-BM28-C (0.15 mg/ml), anti-BM28-P (0.25 mg/ml), and anti-GST (1 mg/ml) antibodies were coinjected with the pHIV2-LTR-CAT-556/+156 and pSPVA1 plasmids as indicated. Undigested HIV and VA probes marked P (10% of total) are shown in lane 8. Readthrough (RT) and terminated (TM) transcripts and VA1 Pol III transcripts are indicated. The results were quantified as in panel A. Size markers in the left-hand lane are MspI-digested pBR322, from 404 to 67 bases. (D) Southern blot of pSPVA1 and pHIV2-LTR plasmids recovered from injected oocytes. One oocyte equivalent of the samples, analyzed in panel C, lanes 3 to 7, was RNase treated, electrophoresed on an agarose gel, blotted, and hybridized to RNA probes complementary to pSPVA1 and pHIV2-LTR-CAT-556/+156. Lanes 1 and 2 were loaded with a mixture of uninjected supercoiled plasmids (U).

FIG. 2

MCM2 and MCM3 copurify with Xenopus oocyte Pol II holoenzyme. Affinity columns (250 μl) containing GST or GST-TFIIS at 10 mg/ml were loaded in parallel with 50 mg (2 ml) of X. laevis oocyte extract in the presence of ethidium bromide, washed five times with 1 ml of CB plus 50 mM NaCl, and eluted with 1.2 ml of CB plus 0.325 mM NaCl. A total of 0.25% of the load and the flowthrough (FT), 8% of the final wash, and 10% of the eluate fractions were analyzed by Western blotting with the indicated antibodies.

FIG. 3

HeLa MCMs and Pol II holoenzyme components bind to VP16 and TFIIS affinity columns. Affinity columns (1 ml) containing GST (10 mg/ml), GST-VP16 (6 mg/ml), or GST-TFIIS (10 mg/ml) were loaded in parallel with 240 mg of HeLa whole-cell extract, washed five times with 3 ml of CB plus 50 mM NaCl, and eluted with 5 ml of CB plus 0.325 mM NaCl. A total of 0.025% of the load (L) and the flowthrough (FT) fractions and 0.5% of the final wash (W) and eluate (E) fractions were analyzed by Western blotting with the indicated antibodies. The data are representative of five independent experiments.

FIG. 4

TFIIS-bound MCMs and Pol II holoenzyme comigrate on a gel filtration column. The holoenzyme fraction from a GST-TFIIS affinity column (0.325 M NaCl eluate) was fractionated on a Sepharose CL-2B column. A total of 0.5% of the load (L) and 5% of each fraction (fraction numbers are at the top of each lane) were analyzed by Western blotting with the indicated antibodies. The migrations of dextran blue 2000 (2-MDa) and thyroglobulin (660-kDa) mass markers are indicated. This experiment is representative of four independent fractionations.

FIG. 5

Copurification of Pol II holoenzyme and MCMs by cation-exchange chromatography, sucrose gradient sedimentation, and TFIIS affinity chromatography. (A) HeLa whole-cell extract was fractionated on Biorex 70, and the 0.3 to 0.6 M K acetate fraction (L) was separated on a 10 to 60% sucrose gradient (see Materials and Methods). The Load (L) and alternate fractions from the gradient were analyzed by Western blotting with the indicated antibodies. Note the comigration of Pol II with MCM2 and -3. (B) Pol II-containing sucrose gradient fractions (15 to 20) were chromatographed on GST and GST-TFIIS affinity columns (125 μl) in the presence of ethidium bromide. The columns were washed five times with 0.5 ml CB plus 50 mM NaCl and eluted with CB plus 0.325 M NaCl and CB plus 1 M NaCl. A total of 2.5% of the load (L) and the flowthrough (FT) fractions, 12% of the final wash (W), and 10% of the 0.325 and 1 M NaCl eluates were analyzed by Western blotting with the indicated antibodies.

FIG. 6

Coimmunopurification of Pol II holoenzyme and MCMs with anti-MCM3 antibody. Anti-MCM3 and control rabbit IgG immunoaffinity columns were loaded with HeLa whole-cell extract, washed, and eluted with 1 M NaCl. A total of 0.25% of the load (L) and flowthrough (FT) and 10% of the final wash (W) and eluate (E) fractions were analyzed by Western blotting with the indicated antibodies (Ab).

FIG. 7

Anti-CTD antibody disrupts association of MCMs with the holoenzyme. (A) Diagram of the antibody disruption experiment. GST-TFIIS and GST control columns (100 μl) were loaded with HeLa extract and washed with CB plus 50 mM NaCl. Control (anti-myc; 9E10) or anti-CTD (8WG16) antibody (6 μg in 50 μl of CB plus 50 mM NaCl) was added to the GST-TFIIS resins. The GST column was eluted with buffer only. After 1 h, the antibody eluates were collected and the columns were washed and eluted with CB plus 0.325 M NaCl. (B) Western blots of holoenzyme components displaced by anti-CTD antibody. A total of 0.25% of the load (L), 16% of the control (α-myc) and anti-CTD antibody eluates, and 20% of the high-salt eluates (E) were analyzed with the indicated antibodies. The buffer eluate of the control GST column is shown in lane 2.

FIG. 8

MCM proteins bind to recombinant CTD. HeLa nuclear extract was chromatographed on GST (lane 2), GST-mutant CTD (mut; lane 3), and GST–wild-type CTD (wt; lane 4) affinity resins. Western blots with the indicated antibodies of 0.05% of the load and 1% of the eluates are shown.