Figure 1.
TFII-I is a novel Pol ζ-binding protein.
(A) TAP-Rev7 complexes from 293T cells without (−) or with UV-C (254 nm) irradiation (10 J/m2) were purified in separate experiments, analyzed with SDS-PAGE, and stained with Coomassie. The doublet bands belonging to TFII-I are labeled. Note that cleaved TAP-Rev7 co-migrated with a contaminating band at 28 kDa. (B) Lysates of parental 293T cells (without TAP-Rev7 expression) and the IgG or anti-TFII-I immunoprecipitates (IP) were blotted with anti-TFII-I or anti-Rev7 antibodies. (C) 35S-TFII-I in rabbit reticulocyte lysate (Input) or bound to GST or GST-Rev7 beads were separated by SDS-PAGE and analyzed with a phosphor imager (top panel) or stained with Coomassie (bottom panel). (D) Schematic drawing of TFII-I and its fragments. The six TFII-I repeats R1-R6 are indicated. The Rev7-binding activities of various TFII-I fragments are shown on the right. (E) Purified recombinant TFII-I (residues 350–667), the Rev7 R124A–Rev3L (residues 1847–1898) complex, and Rev1 CTD were mixed at 1∶1∶1 molar ratios and fractioned on a Superdex 200 column. The UV traces of the complex (red) and the molecular mass standards (in blue) are shown. The calculated native molecular mass of the complex is indicated. (F) The indicated column fractions in (E) were separated by SDS-PAGE and stained with Coomassie. The eluting positions of the native molecular mass standards are indicated by arrowheads.
Figure 2.
TFII-I depletion in human cells causes UV and cisplatin sensitivity.
(A) Lysates of 293T cells transfected with the indicated siRNAs were blotted with indicated antibodies. (B) Quantitative PCR analysis of the Rev3L mRNA levels in mock or siRev3L transfected 293T cells. (C) Colony survival curves of 293T cells that were transfected with the indicated siRNAs and irradiated with increasing doses of UV. The mean and standard deviation (SD) of three independent experiments are shown. (D) Colony survival curves of 293T cells that were transfected with the indicated siRNAs and increasing doses of cisplatin. The mean and standard deviation (SD) of three independent experiments are shown. (E) Colony survival curves of U2OS cells that were transfected with the indicated siRNAs and irradiated with increasing doses of UV. The mean and standard deviation (SD) of three independent experiments are shown. (F) Colony survival curves of U2OS cells that were transfected with the indicated siRNAs and increasing doses of cisplatin. The mean and standard deviation (SD) of three independent experiments are shown.
Figure 3.
TFII-I is required for DNA damage tolerance in human cells.
(A) HeLa Tet-On cells were transfected with the indicated siRNAs and plasmids, treated with UV (10 J/m2), and harvested at various timepoints for flow cytometry. The percentage of γ-H2AX-positive cells was plotted against the time after UV irradiation. The means and SDs of two experiments are shown. (B) Representative dot plots of the flow cytometry analysis of cells in (A). (C) Lysates of cells in (A) were blotted with anti-TFII-I and anti-tubulin antibodies. (D) HeLa Tet-On cells were either mock transfected or transfected with siTFII-I along with a control vector or the Myc-TFII-I plasmid and irradiated with UV (10 J/m2). At 24 h after UV treatment, cells were fixed and stained with DAPI (blue) and anti-γ-H2AX antibody (red).
Figure 4.
TFII-I is required for DNA translesion synthesis (TLS) in human cells.
(A) The mutation frequency of UV-treated SupF plasmid in 293T cells transfected with the indicated siRNAs. The mean and SD of three experiments are shown. (B) The mutation frequency of UV-treated SupF plasmid in 293T cells transfected with the indicated siRNAs. The mean and SD of two experiments are shown. (C) The mutation spectra of the UV-irradiated SupF gene recovered from 293T cells transfected with the indicated siRNAs. The number of clones sequenced for each group is shown in parentheses.
Figure 5.
Depletion of TFII-I or Rev7 does not grossly impact transcription.
(A) Quantitative PCR analysis of the mRNA levels of Rev1, Rev7, Rev3L, and Rad18 in mock or siTFII-I transfected 293T cells. The mean and standard deviation of triplicate samples are shown. (B) Venn diagram of genes whose expression was down 2-fold or more in HeLa Tet-On cells depleted of TFII-I or Rev7, as determined by microarrays. The bold text highlights the genes whose expression is affected in both sets of cells. GTF2IP1 and Mad2L2 encode TFII-I and Rev7, respectively, and are underlined. Their mRNAs are expected to be depleted by the siRNAs, and serve as positive controls.
Figure 6.
The PCNA-binding motifs and N-terminal region of TFII-I are required for translesion synthesis.
(A) Schematic drawing of the domains and motifs of human TFII-I γ. LZ, leucine zipper. (B) U2OS cells were transfected with siControl or siTFII-I along with the indicated TFII-I plasmids, irradiated with UV (60 J/m2), and treated with formaldehyde. Lysates, anti-TFII-I IP, and anti-PCNA IP of these cells were blotted with the indicated antibodies. (C) U2OS cells were transfected with GFP-TFII-I wild type (WT) or mAPIM and DsRed-PCNA plasmids, and micro-irradiated with a 365-nm laser along straight lines. GFP and DsRed channels are shown separately in gray scale and together in merge images. Scale bars, 10 µm. (D) The mutation frequency of the UV-treated SupF plasmid in 293T cells transfected with the indicated siRNAs and plasmids. The mean and SD of six experiments are shown.
Figure 7.
The TFII-I dimer bridges PCNA and Rev7.
(A) The monomeric TFII-I fragment (residues 330–667) does not form a ternary complex with PCNA and Rev7. The indicated combinations of recombinant purified Rev7 R124A, PCNA, and TFII-I (residues 330–667) proteins were fractionated on a Superdex 200 gel filtration column. Selected fractions were separated by SDS-PAGE followed by Coomassie staining. The positions of native molecular mass standards are indicated by arrowheads. (B) Purified TFII-I1–667 and TFII-I270–667 fragments were fractionated on a Superdex 200 gel filtration column. (C) HeLa Tet-On cells were transfected with the indicated plasmids. Lysates, IgG IP, and anti-Myc IP of these cells were blotted with the indicated antibodies. (D) Model for TFII-I-dependent recruitment of Pol ζ (Rev3L–Rev7) to PCNA at DNA damage sites during translesion synthesis. Ub, ubiquitin. CTD, C-terminal domain.