The minichromosome maintenance proteins 2-7 (MCM2-7) are necessary for RNA polymerase II (Pol II)-mediated transcription

Abstract

The MCM2-7 (minichromosome maintenance) proteins are a family of evolutionarily highly conserved proteins. They are essential for DNA replication in yeast and are considered to function as DNA helicases. However, it has long been shown that there is an overabundance of the MCM2-7 proteins when compared with the number of DNA replication origins in chromatin. It has been suggested that the MCM2-7 proteins may function in other biological processes that require the unwinding of the DNA helix. In this report, we show that RNA polymerase II (Pol II)-mediated transcription is dependent on MCM5 and MCM2 proteins. Furthermore, the MCM2-7 proteins are co-localized with RNA Pol II on chromatins of constitutively transcribing genes, and MCM5 is required for transcription elongation of RNA Pol II. Finally, we demonstrate that the integrity of the MCM2-7 hexamer complex and the DNA helicase domain in MCM5 are essential for the process of transcription.

FIGURE 1.

MCM5 and MCM2 are essential for general transcription. A, 293T cells were left untreated or transfected with either a negative control siRNA or MCM5 siRNA. Whole cell extracts were prepared after 4 days, and Western blot analysis was performed with anti-MCM5, anti-MCM3, anti-Stat1, and anti-Stat3 antibodies. B, 293T cells transfected as described in A were treated with 50 μm [5,6-H-3]uridine 40–50 Ci/mmol for 5, 20, and 120 min. Cells were washed once with 1× phosphate-buffered saline and resuspended in 500 μl of 10% trichloroacetic acid. The precipitated nucleotides were collected with glass microfiber filters, and cpm were determined with a scintillation counter. Data represent the averages and standard deviations (error bars) of three experiments. C, 293T cells were transfected with either a negative control siRNA or MCM2 siRNA and cultured for 3 days, and Western blot was performed with anti-MCM2, anti-MCM5, and anti-Stat3 antibodies. D, cells transfected as described in C were analyzed by tritium incorporation assays as described in B.

FIGURE 2.

Cell cycle analyses of cells with MCM5 knockdown by siRNA. 293T cells were transfected as described in the legend for Fig. 1. Cells were cultured for 4 days, and 1 × 10⁶ cells were stained with propidium iodide (PI) and analyzed by FACs.

FIGURE 3.

MCM5 is essential for RNA Pol II-mediated transcription. A, 293T cells transfected as in Fig. 1A were grown for 4 days, scraped, and washed with 1× phosphate-buffered saline. Nuclear run-on assays were performed with slot-blot membranes containing 5 μg of plasmid DNA or poly(A) RNA. CBP, CREB-binding protein. B, results from A were quantitated with a PhosphorImager (Amersham Biosciences). Results represent one of two independent experiments.

FIGURE 4.

Members of the MCM2-7 family co-localize with RNA Pol II at the loci of constitutively transcribed genes. A, map of the GAPDH gene and primers used for ChIP analysis against the 5′ region and the center (C) of the gene. B and C, 2fTGH cells were grown in 15-cm dishes to about 80–90% confluence. ChIP assays were performed using anti-MCM3, anti-MCM5, and anti-RNA Pol II antibodies. PCR reactions used primers against the 5′ region or the center of the GAPDH gene. C, results were quantitated using a PhosphorImager and expressed as ChIP/Input. D, ChIP assays were performed for 2fTGH cells with anti-Stat1, anti-MCM2, anti-MCM3, anti-MCM5, and anti-RNA Pol II antibodies. One 15-cm dish was used for the Stat1, MCM2, and MCM3 ChIP (lanes 1–3), and a second dish was used for the MCM5, RNA Pol II (lanes 4 and 5), and Stat1 ChIP. The result of one Stat1 IP (lane 1) is shown. Input A (lane 6) corresponds to the Stat1, MCM2, and MCM3 ChIP (lanes 1–3), and Input B (lane 7) corresponds to Mcm5 and RNA Pol II (lanes 4 and 5). Primers correspond to the centers of the β-actin, E2F4, and β-tubulin genes. E, signals in D were quantitated with a PhosphorImager, and values are expressed as ChIP/Input. Results represent at least three independent experiments.

FIGURE 5.

MCM5 is required for transcription elongation of RNA Pol II. RNAi-mediated MCM5 knockdown was performed in 293T cells as described in the legend for Fig. 1, and ChIP analysis was performed with RNA Pol II antibody as described in the legend for Fig. 4. A, primers against the 5′ intergenic region (5′), promoter region (P), and center (C) of the GAPDH gene were used for ChIP analysis in both control (Ctl) and MCM5 siRNA-transfected (M5) cells. C, ChIP was performed with primers against the 5′ regions (5′) and centers (C) of E2F4, β-actin, and β-tubulin. B and D, signals from A and C were quantitated with a PhosphorImager and shown as ChIP/INPUT with results from control siRNA cells set as 1.

FIGURE 6.

Requirement of the MCM2-7 hexamer and helicase activity for transcription. A, 293T cells were transfected with either MCM5 siRNA or a negative control siRNA (Contl) and grown for 3 days. Cells were then transfected with pCMV, pMCM5WT, pMCM5KMD, or pMCM5RK and grown for 24 h. Whole cell extracts were prepared from a portion of the cells and analyzed by Western blot analysis. HA, hemagglutinin A (HA). B, the remaining cells were treated with 50 mm [5,6-H-3]uridine 40–50 Ci/mmol for 2 h followed by trichloroacetic acid precipitation and analysis of tritium incorporation. Results represent one of three independent experiments.