A neomorphic cancer cell-specific role of MAGE-A4 in trans-lesion synthesis

Trans-lesion synthesis (TLS) is an important DNA-damage tolerance mechanism that permits ongoing DNA synthesis in cells harbouring damaged genomes. The E3 ubiquitin ligase RAD18 activates TLS by promoting recruitment of Y-family DNA polymerases to sites of DNA-damage-induced replication fork stalling. Here we identify the cancer/testes antigen melanoma antigen-A4 (MAGE-A4) as a tumour cell-specific RAD18-binding partner and an activator of TLS. MAGE-A4 depletion from MAGE-A4-expressing cancer cells destabilizes RAD18. Conversely, ectopic expression of MAGE-A4 (in cell lines lacking endogenous MAGE-A4) promotes RAD18 stability. DNA-damage-induced mono-ubiquitination of the RAD18 substrate PCNA is attenuated by MAGE-A4 silencing. MAGE-A4-depleted cells fail to resume DNA synthesis normally following ultraviolet irradiation and accumulate γH2AX, thereby recapitulating major hallmarks of TLS deficiency. Taken together, these results demonstrate a mechanism by which reprogramming of ubiquitin signalling in cancer cells can influence DNA damage tolerance and probably contribute to an altered genomic landscape.

were added to give a concentration of 20 g/ml and incubations were continued for 1 h. Finally 3 l of glutathione donor beads (Perkin Elmer 6765300) were added (20 g/ml final concentration) and incubations were continued for one more hour. Plates were analyzed using the Perkin Elmer Envision plate reader to excite at 680 nm and detect fluorescence at 520 nm.
(b) The ALPHAscreen assay was used to detect the association of GST-RAD18 335-400 with His-tagged full-length MAGE-A4, exactly as described for RAD18-RAD6 association in (a) above. Note that the binding curves in (a) and (b) cannot be compared directly because in ALPHAscreen assays the magnitude of signal for each unique protein-protein interaction is determined by specific protein conformations and tag proximities.
(c) ALPHAscreen assay was used to detect competition between RAD6 and MAGE-A4 for RAD18-binding. In separate reactions His-RAD6 (25 nM) or His-MAGE-A4 (200 nM) were incubated with equimolar GST-RAD18 in the presence of different concentrations (0-200 nM) of untagged competitor RAD6 (or BSA for control). Donor and acceptor beads were added to the reactions and plates were analyzed as described in (a) above. To plot the data, the effect of untagged RAD6 (or BSA) on fluorescence was normalized to emission signals obtained in the RAD18/His-RAD6 and RAD18/His-MAGE-A4 reactions without added protein. Error bars represent the mean of 16 replicate wells +/-SEM for each experimental condition.
(d) Effect of MAGE-A4 expression on RAD18-associated RAD6 in cultured cells. 293T cells were transfected with expression vectors encoding MYC-RAD18 and MAGE-A4 (or empty vector for control). Cell lysates were prepared 48 h after transfection, and RAD18 was immunoprecipitated using anti-MYC beads. Levels of RAD18-associated RAD6 were determined by SDS-PAGE and immunoblotting. Note that in this experiment, levels of ectopically-expressed RAD18 were very high and no longer sensitive to co-expressed MAGE-A4.
(e)-(h) Size fractionation of RAD18, RAD6 and MAGE-A4 complexes in cultured cells. Exponentially-growing cultures of 293T cells were transfected with empty vector, CMV-GFP, or with a CMV-MAGE-A4 expression plasmid. 48 h later GFP fluorescence was used to confirm efficient (~90%) transfection efficiency. Cells were lysed in 300 l of CSK without sucrose and supplemented with 1 g/ml (25,000 units/ml) of Benzonase. Lysates were incubated at room temperature for 15 minutes to digest chromatin, then centrifuged at 21,000g for 20 min. 250 l of each clarified cell lysate (~2.0 mg) was loaded onto a 25ml Sephadex 200 gel filtration column that was calibrated with appropriate molecular weight markers (e). The column was eluted with sucrose-free CSK. 0.5 ml fractions were collected and 30 l of each fraction was analyzed by SDS-PAGE and immunoblotting with antibodies against RAD18, RAD6 and MAGE-A4 (f). Relative levels of RAD18, RAD6 and MAGE-A4 between different fractions were determined by densitometry and are plotted in panels (g) and (h). The fractions containing the ~160 kD [RAD18] 2 -RAD6 complex predicted by biophysical studies and a previously undescribed ~530 kD RAD18 and RAD6-containing complex are indicated.

Supplementary Fig. 2 Quantitative immunoblot analysis of RAD18, RAD6 and MAGE-A4 expression levels in H1299 cells.
The indicated amounts of H1299 whole cell lysate, recombinant RAD18-RAD6 (a), and MAGE-A4 (b) proteins were analyzed by SDS-PAGE and immunoblotting. By comparing expression levels of proteins in cell lysates with defined quantities of purified protein standards we estimate that 1 g of H1299 cell lysate contains 41 pg (0.74 fmol) of RAD18, 719 pg (42 fmol) of RAD6, and 88 pg (2.5 fmol) of MAGE-A4. Therefore, the RAD18:RAD6:MAGE-A4 stoichiometry in H1299 cells is approximately 1:57:3. Fig. 3c showing UV-induced distribution of RAD18 but not of MAGE-A4 to nuclear foci.

Supplementary Fig. 3 Expanded version of
H1299 cells were transiently transfected with an expression plasmid encoding CFP-RAD18 or with an empty vector for control. Transfected cells were UV-irradiated (20 J/m 2 ) or were left untreated. After 6 h, cells were fixed and stained with antibodies against MAGE-A4. The subcellular distribution of MAGE-A4 and CFP-RAD18 was visualized using immunofluorescence microscopy. Scalebar = 10 m.