Fig 2: 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.

Fig 3: 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.

Fig 4: 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.

Fig 5: 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.