PMC

In vitro far-Western assay verifying the interactions between polι UBM1 (F507S and P511R) and UBM2 (P680A) substitutions with polη, PCNA, ubiquitin and K63-linked ubiquitin chains. Purified His-tagged wild-type polι and the indicated mutants were separated by SDS–PAGE, transferred to nitrocellulose and incubated with in vitro translated ³⁵S-labeled polη (A), PCNA (B) and ubiquitin (C). Densitometric analysis of far-Westerns (top panels) compares the strength of interaction with wild-type polι and mutants with the ³⁵S-labeled proteins; the ³⁵S band intensities (middle panels) were normalized to their respective Ponceau-stained bands (bottom panels). (D) Wild-type polι and UBM mutants interact with K63-linked Ub chains; 15 µg of K63-linked ubiquitin chains (Boston Biochem) were separated by SDS–PAGE, transferred onto nitrocellulose and incubated with ³⁵S-labeled wild-type polι or the indicated UBM mutant.

Polι UBM1 mutants (F507S and P511R) do not localize into DNA damage-induced foci. MRC5 human cells were transfected with plasmids encoding eCFP-polι wild type (peCFP-C1-polι), eCFP-polι_F507S (pJRM23) and eCFP-polι_P511R (pMGB9). Twenty hours after post-transfection, the cells were irradiated with UV (7 J/m²). After 6 h, cells were fixed and the presence of foci was examined. The histogram represents the mean number of cells with foci. Error bars are the standard deviation calculated after counting 200 cells in 2–5 independent experiments with each construct.

Analysis of the polη UBZ residues that are responsible for the interaction with polι. (A) Structure of the human polη UBZ domain (PDB: 2I5O) with key residues used in the analysis are highlighted. Zn²⁺ is indicated as a bronze sphere. (B) Yeast two-hybrid assay showing the effect of mutating polη UBZ residues on their ability to interact with polι. Wild-type polι is unable to interact with polη C635A, C638A and D652A substitutions, whereas the polι P692L substitution facilitates an interaction with the various UBZ mutants. Images were taken after 4 days of incubation at 30°C.

Cartoon explaining how the various interactions between polι, polη and PCNA can be modulated by ubiquitin. The polymerases are indicated as a rod with functional domains/motifs colored as follows: catalytic domain of polι, light blue; catalytic domain of polη, dark blue; PIP-box, purple rectangle; PCNA, purple disk; wild-type UBM1/UBM2/UBZ, green rectangle; mutant UBM1/2, red rectangle; wild-type ubiquitin, orange ellipsoid; I44A Ubiquitin mutant, red ellipsoid. (A) polι interacts with ubiquitinated polη predominantly via UBM2. Polι can still bind PCNA via is PIP-box, but ubiquitinated polη is unable to bind PCNA (); (B) when UBM2 is unavailable, polι can potentially interact with ubiquitinated polη via UBM1; (C) polι cannot interact with ubiquitinated polη when both UBMs are mutated; (D) mutation of polη’s natural ubiquitination sites blocks the interaction between polη and polι; (E) the polι-Ub chimera binds to the UBZ of polη. Both polymerases are able to interact with PCNA; (F) the I44A mutation in the polι-Ub chimera inhibits the interaction between polι and polη, but allows for an interaction between ubiquitin and UBM2.

Interaction between polι and polη depends on polη ubiquitination. Yeast two-hybrid analysis of interactions between polη carrying four lysine point mutations (K682A, K686A, K694A and K709A) and polι, PCNA and ubiquitin. Yeast strain AH109 was transformed separately with the GAL4-AD expression vectors pACT2, pACT2-polι wild type (pAR116) and pACT2-PCNA (pAVR17) and pACT2-Ub (pBP127) in combination with each one of the following GAL4-BD expression vectors: pGBKT7 and pGBKT7-polη wild type (pAVR65), pGBKT7-polη 4K→A (pMGB4) as indicated. Several colonies from each transformation were grown overnight at 30°C in selective medium, and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 30°C for 6 days. The polη 4K→A mutant is unable to interact with polι, suggesting that the interaction is between polι and monoubiquitinated polη.

Mapping the region in polη that interacts with polι using a yeast two-hybrid assay. (A) Cartoon of polη deletion constructs. The dark gray rectangle is the catalytic core of polη, the UBZ motif is indicated as a gray diamond and the PCNA-interacting motif (PIP-box) is indicated as a light gray box. (B) Yeast two-hybrid assay showing the interaction between full-length polι and deletion alleles of polη. Deletion mapping reveals that the interaction with polι is localized to a region containing the UBZ domain of polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pACT2-polι wild type (pAR116) and (I) pGBKT7-polη wild type (pAVR65), or (II) pGBKT7-polη_Δ1-483 (pAVR45), or (III) pGBKT7-polη_Δ484-587 (pAVR51), or (IV) pGBKT7-polη_Δ587-641(pAVR52). Images were taken after 4 days of incubation at 30°C.

Polι interacts with polη through its UBM domains. Ribbon diagrams show the structure of the UBMs (green) interacting with Ubiquitin (bronze). (A) Localization of UBM1 mutants. The model of human UBM1 was generated based upon the closely related murine UBM1 structure (PDB 2KWV). Residues that impair the interaction with polη are highlighted in purple. (B) Localization of UBM2 mutants. The human UBM2-ubiquitin structure was generated using PDB 2KHW. Residues that simultaneously disrupt the interaction with ubiquitin and polη are highlighted in yellow. Residues that do not impair the interaction with ubiquitin or polη are highlighted in blue. The P692 residue, which when changed to Arg selectively disrupts the interaction with ubiquitin, is highlighted in red. (C) A two-hybrid assay demonstrating that the F507S/P692R UBM1-UBM2 double mutant does not interact with polη or ubiquitin, whilst retaining its ability to interact with PCNA. Yeast strain AH109 was transformed separately with the GAL4-AD expression vectors pACT2, pACT2-polι wild type (pAR116) and pACT2-polι F507S/P692R (pJRM142) in combination with one of the following GAL4-BD expression vectors: pGBKT7, pGBKT7-polη wild type (pAVR65), pGBKT7-PCNA (pAVR18) and pGBKT7-Ub (pBP129) as indicated. Several colonies from each transformation were grown overnight at 30°C in selective medium, and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 30°C for 6 days.

Analysis of polι UBM1 and UBM2 residues that are responsible for the interaction with polη. (A) Sequence alignment of UBM1 and (B) UBM2. Conserved residues mutated in the analysis are shaded gray. The aligned polι proteins are from the following mammals: Hs, Homo sapiens; Mm, Mus musculus; Cl, Canis lupus; Mf, Macaca fascicularis; Bt, Bos taurus; Rn, Rattus norvegicus. (C) Yeast two-hybrid analysis of the interactions between polι UBM1 and UBM2 mutants and polη, PCNA and ubiquitin (Ub). Saccharomyces cerevisiae strain AH109 was transformed separately with the GAL4-AD expression vectors pACT2 (control), pACT2-polι wild type (pAR116), pACT2-polι carrying various point mutations in UBM1 or UBM2 as indicated in combination with each one of the following GAL4-BD expression vectors: pGBKT7 and pGBKT7-polη_wild type (pAVR65), pGBKT7-PCNA (pAVR18) and pGBKT7-Ub (pBP129) as indicated. Several colonies from each transformation were grown overnight at 30°C in selective medium, and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 30°C for 6 days. Four and six represent days of growth at 30°C.

Interactions between a polι-Ub chimera and polη. (A) Cartoon of the polι-Ub chimera with the I44A substitution indicated. Yeast two-hybrid analysis of interactions between polι, polι-Ub, polι-Ub-I44A, and polι-F507S-P680A-Ub and wild-type polη, PCNA and ubiquitin (Ub). Saccharomyces cerevisiae strain AH109 was transformed separately with the GAL4-AD expression vectors pACT2 (control), pACT2-polι wild type (pAR116), pACT2-polι-Ub (pJRM127), polι-Ub_I44A (pJRM150) and polι-F507S/P680A-Ub (pJRM151) as indicated, in combination with each of the following GAL4-BD expression vectors: pGBKT7 and pGBKT7-polη_wild type, (pAVR65), pGBKT7-PCNA (pAVR18) and pGBKT7-Ub (pBP129) as indicated. Several colonies from each transformation were grown overnight at 30°C in selective medium, and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 30°C for 4 days. Images were taken after 2 days of growth () or 4 days of growth (). (B) FLAG-pull-down assay demonstrating interactions between polι and polη (lane 2), and polι-Ub and polη (lane 4). Extracts from HEK293T cells transfected with plasmids encoding FLAG-tagged wild-type polι (pJRM46) or a polι-Ub fusion (pJRM140) and HA-tagged wild-type polη (pJRM56) were incubated overnight at 4°C with 20 µl of EZview Red ANTI-FLAG M2 Affinity Gel, washed three times and analyzed directly by SDS–PAGE and Western blot with respective antibodies. Lanes 1 and 3 represent 10% of corresponding extracts used for each pull-down reaction. (C) FLAG-pull-down assay demonstrating the strength of interactions between PCNA and polι (lane 2), or polι-Ub (lane 4). Extracts from HEK293T cells transfected with plasmids encoding FLAG-tagged wild-type polι (pJRM46) or a polι-Ub fusion (pJRM140) were incubated overnight at 4°C with 20 µl of EZview Red ANTI-FLAG M2 Affinity Gel, washed three times and analyzed directly by SDS–PAGE and Western blot with respective antibodies. Lanes 1 and 3 represent 10% of corresponding extracts used for each pull-down reaction. (D) MRC5 human cells were transfected with plasmids encoding eCFP-polι wild type (peCFP-C1-polι) and eCFP-polι-Ub (pJRM128). Twenty hours after post-transfection, the cells were irradiated with UV (7 J/m²). After 6 h, cells were fixed and the presence of foci examined. The histogram represents the mean and standard deviation calculated after counting 200 cells from three independent experiments with each construct.