Volume 39, Issue 4, Pages 632-640 (August 2010) MMXD, a TFIIH-Independent XPD-MMS19 Protein Complex Involved in Chromosome Segregation  Shinsuke Ito, Li Jing Tan, Daisuke Andoh, Takashi Narita, Mineaki Seki, Yasuhiro Hirano, Keiko Narita, Isao Kuraoka, Yasushi Hiraoka, Kiyoji Tanaka  Molecular Cell  Volume 39, Issue 4, Pages 632-640 (August 2010) DOI: 10.1016/j.molcel.2010.07.029 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 39, 632-640DOI: (10.1016/j.molcel.2010.07.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 XPD and MMS19 Are Included in a Protein Complex (A) Experimental procedure for purification of either the XPD or MMS19 complex. (B) Silver staining of the XPD and MMS19 complexes purified from whole-cell extracts. As a control, a mock purification was performed with whole-cell extract from nontransfected HEK293 cells. Purified polypeptides were processed for mass spectrometry. Note that, in these purifications, ANT2 was clearly detected in both complexes. Polypeptides in mock lane are contaminants, which are marked with an asterisk. (C) Whole-cell lysate and the purified XPD and MMS19 complexes were immunoblotted with antibodies against indicated proteins. The tagged proteins are slightly larger than the endogenous ones. (D) The MMS19 complex was further separated on a Superose 6 column. After fractionation, each fraction was resolved by SDS-PAGE, visualized by silver staining (top), and immunoblotted with indicated antibodies (bottom). Estimated molecular sizes are depicted in the top panel. Molecular Cell 2010 39, 632-640DOI: (10.1016/j.molcel.2010.07.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 XPD Directly Interacts with MMS19 and MIP18 (A) Immunoblot of whole-cell extract from HeLa cells transfected with control siRNA (siCONT), siRNA for MMS19 (siMMS19), or siRNA for MIP18 (siMIP18) using the indicated antibodies. (B) Real-time quantitative PCR analysis was performed with cDNA prepared from siCONT-, siMMS19-, or siMIP18-transfected HeLa cells. The histograms represent the mean value of MMS19, MIP18, XPD, and actin mRNAs normalized with GAPDH mRNA. Error bars indicate SD calculated from three independent experiments. (C) GST or GST-fused MIP18 was expressed in bacteria, immobilized on glutathione Sepharose beads, and incubated with either MMS19 or XPD protein produced in a baculovirus expression system (Figure S3A). The proteins bound to the GST or GST-MIP18 beads were then examined by immunoblotting with anti-MMS19 and anti-XPD antibodies, respectively. 10% of MMS19 or 2% of XPD proteins used for the GST pull-down assay were loaded as input (IN). (D) Schematic representation of XPD domains and regions of XPD polypeptides for binding of MAT1 and p44. Two RecA-like domains (HD1, green; HD2, blue), an Iron-sulfer (FeS) cluster domain (FeS, orange), and an Arch-shaped domain (Arch, gray) are shown. Helicase motifs are shown as red boxes. (E) FLAG-MMS19 (fMMS19) purified from lysate of fMMS19 baculovirus-infected Sf9 cells using anti-FLAG M2-agarose was incubated with full-length or truncated XPD polypeptides synthesized in vitro and labeled with [35S]-methionine. As a control, lysate of noninfected Sf9 cells was used. After a wash with NETN, XPD polypeptides bound to fMMS19 or mock beads as well as the input (IN) were loaded on the polyacrylamide gel and detected by autoradiography. The asterisk indicates a nonspecific band. Molecular Cell 2010 39, 632-640DOI: (10.1016/j.molcel.2010.07.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Colocalization of MIP18 with Mitotic Spindles and MMXD siRNA Knockdown Lead to Abnormal Mitosis and Nuclei in HCT116 Cells (A) Immunofluorescence analysis of mitotic HCT116 colon cancer cells using anti-MIP18, -MMS19, -XPB, or -α-tubulin antibody and of XP-D cells (XP6BE) stably expressing XPD-GFP. Note that strong colocalization of MIP18 and partial colocalization of MMS19 and XPD-GFP with the mitotic spindles was detected. On the other hand, no colocalization of XPB was detected. Scale bar, 10 μm. (B) Representative figures of normal mitotic spindles in control siRNA-treated HCT116 cells (siCONT) and abnormal monopolar and multipolar spindles with misaligned chromosomes in siMMS19-, siMIP18-, and siXPD-transfected HCT116 cells. Cells were stained with anti-α-tubulin (green), siRNA labeled with cy3 (red), and DAPI (blue) for DNA, respectively. Scale bar, 10 μm. (C) The number of cells with abnormal mitotic spindles was scored using a fluorescence microscope for siCONT-, siMMS19-, siMIP18-, siXPD- and siXPB-transfected HCT116 cells. Histograms show average frequency of abnormal mitotic spindles, and error bars indicate SEM from three independent experiments. More than 150 mitotic cells were counted for each siRNA sample. (D) Abnormal nuclear morphology in siMMS19-, siMIP18-, and siXPD- or siXPB-transfected HCT116 cells, compared with control siRNA-treated (siCONT) cells. Cells were stained with DAPI and merged with siRNAs-cy3. Scale bar, 5 μm. (E) Histograms show average frequency of abnormal nuclei, and error bars indicate SEM from three independent experiments. More than 1500 nuclei were counted for each siRNA sample. Molecular Cell 2010 39, 632-640DOI: (10.1016/j.molcel.2010.07.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Abnormal Mitosis and Nuclei in the Cells Derived from Patients with XPD Mutations and in the XPD cDNA-Corrected Isogenic Cells (A) Example of cells derived from patients with XPD mutations showing abnormal mitotic spindles (monopolar or multipolar with supernumery centrosomes). The cells were stained with γ-tubulin (green), α-tubulin (red), and DAPI (blue). (B) Histograms show the average frequency of abnormal mitotic spindles, and error bars indicate SEM from three independent experiments in the XPD-proficient and -deficient cell lines. At least 150 mitotic cells were scored to calculate the percentage of abnormal mitosis in each cell line. Statistical comparisons were performed by Student's t tests. (C) Cells in interphase were identified by staining with DAPI for DNA. Images represent an abnormal nuclear morphology, for example, abnormal nuclear shape, binuclear, and multinuclear cells derived from patients with XPD mutations. (D) Histograms show the average frequency of aberrant interphase nuclei (binuclear, multinuclear, and abnormal shape). Error bars indicate SEM from three independent experiments. At least 1500 nuclei were scored to calculate the percentage of abnormal nuclei in each cell line. Statistical comparisons were performed by Student's t tests. Molecular Cell 2010 39, 632-640DOI: (10.1016/j.molcel.2010.07.029) Copyright © 2010 Elsevier Inc. Terms and Conditions