Fig. 2. Co-localization of polη and polι. (A) MRC5 cells co-transfected with eGFP–polη and pCDNA-polι were irradiated (7 J/m²), fixed 12 h later and stained with anti-polι antibody and TRITC-conjugated secondary antibody. The staining pattern of polι (red) (left) and the autofluorescent signal of GFP–polη (green) (middle) in the same cell are shown. Co-localization of polι and eGFP–polη is indicated by a yellow pattern (right). (B–D) MRC5 cells were co-injected with peYFP-polη and peCFP-polι, and UV irradiated (10 J/m²) 8 h later. The cells were examined by time-lapse microscopy using appropriate filters. (B) The fluorescent signals for the two tagged proteins at various times after irradiation. In (C), the co-ordinates of each focus in the cell were determined at different times. Each track represents the movement of a specific focus of polη (green) and polι (red). The dashed line represents the approximate position of the nuclear envelope. In (D), each of the four panels represents the intensity of the yellow and blue signals of an individual focus as a function of time. Green, polη; red, polι.

Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. (A) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. (B) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). (C) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained SDS–polyacrylamide gel showing the expression and expected size of the GST–polι fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with ³⁵S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). (D) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated ³⁵S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated ³⁵S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). (E) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His₆-polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.

Fig. 6. Interactions between pol η and polι, and involvement in TLS. (A) Domain structures of polη and polι. FF, domains required for foci formation; NLS, classical nuclear localization signal; NLD, nuclear localization domain. The black bar indicates regions of interaction between the two polymerases. (B) Involvement of polymerases in bypass of damage. (1) The structure shows a replication fork attached to a nuclear matrix structure in a replication factory, with the DNA being pulled through the fork. Polη and polι are either co-localized in the factory and get recruited to the replisome or, in (2), they are an intrinsic part of the replisome, possibly by interacting with PCNA. (3) When polδ is blocked by damage, it either disengages from the primer terminus or is removed following modification, allowing polη or polι to bypass the damage. In (4), polδ is restored to the replication fork after the damage has been bypassed. (C) Scheme for interaction of polymerases. The depicted N-terminal catalytic domain is drawn in schematic form from the crystal structure of a Y-family DNA polymerase from Sulfolobus solfataricus (Ling et al., 2001), with the finger, palm, thumb and little finger domains indicated in blue, red, green and purple, respectively.

Fig. 1. Polι does not correct the UV + caffeine sensitivity of XP-V cells but localizes into nuclear foci after UV irradiation. (A) XP30RO cells were transfected with the plasmids pCDNA3.1 vector, pCDNA-polι or pCDNA-polη as indicated. Stable clones were UV irradiated (7 J/m²) and then incubated in the presence of 75 µg/ml caffeine. Right panel: MRC5 cells treated with the same doses of UV + caffeine. After 4 days, the cells were stained with methylene blue. (B and C) MRC5 cells were transfected with plasmids encoding either eGFP–polι (B) or pCDNA-polι (C). At 20 h post-transfection, cells were irradiated with 7 J/m² (right panels). After 12 h, the distribution of eGFP–polι was examined following paraformaldehyde fixation. For cells transfected with pCDNA-polι (C), polι distribution was revealed with anti-polι antibody and tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody. (D) Western blots of extracts of MRC5 cells irradiated with 7 J/m² and incubated for the indicated times. Blots were probed with anti-polι. In (E), MRC5 cells were transfected with eGFP–polι and irradiated either uniformly (left) or through a Millipore filter (right).

Fig. 3. Dependence of polι foci on polη. MRC5 (left) or XP30RO cells (right) transfected with eGFP–polι were UV irradiated, fixed 12 h later and stained with anti-PCNA monoclonal and TRITC-conjugated secondary antibody. The staining patterns of the autofluorescent signal of GFP (green staining in upper panels) and PCNA (red staining in lower panels) in the same cell are shown.

Fig. 5. Deletion analysis of polι localization. Polι deletion constructs were made as described in Materials and methods. The fragments were cloned downstream of the eGFP tag and transfected into MRC5 cells. The cells were UV irradiated (7 J/m²) 24 h later and, after a further 8 h, the cells were fixed and analysed for nuclear location and focus formation as indicated.