Our genetic material is highly susceptible to mutations, either spontaneously or triggered by external stimuli such as UV and chemical carcinogens. Human cells employ an extensive array of machineries to repair various types of DNA damage. One such machinery deals with the UV-induced bulky lesions formed in DNA. Normal DNA replication machinery stalls at the sites of DNA lesions. In the absence of a repair machinery intervening to replace the normal DNA replicase with a damage-tolerant replicase, the cell will undergo apoptosis, a form of cell death. This damage-tolerant replicase carries out the replication past the damaged DNA site in a process termed trans-lesion DNA synthesis.
RAD18 is a highly conserved protein that is crucial for trans-lesion DNA synthesis. At the enzymatic level, RAD18 functions as a ubiquitin ligase by attaching a small polypeptide ubiquitin to its substrates. The main substrate of RAD18 is the DNA sliding clamp PCNA, which is part of the replicative machinery. RAD18-mediated ubiquitination of PCNA triggers the polymerase exchange event that is central to trans-lesion DNA synthesis response. Recently, it was found that a Melanoma associated antigen-4 (MAGE-A4), which is selectively expressed in a number of cancers, binds and stabilizes RAD18 [1]. Stabilization of RAD18 has important consequences for cancer cells, especially when cancers are being treated with genotoxic radio or chemo therapeutics. Higher RAD18 levels are known to result in the resistance of multiple cancers to the genotoxicity-inducing chemo/radio therapies [2]. Humans contain ~40 Melanoma associated antigen (MAGE) genes, the majority of which are specifically expressed in cancers and characterized as oncogenes [3]. MAGE proteins have been shown to interact with specific ubiquitin ligases to exert their physiological roles, but the precise nature of this interaction and how it translates into a function is currently being investigated.
The Bhogaraju group at EMBL Grenoble has recently obtained new insights into the mechanism of action of RAD18, and its regulation by MAGE-A4 [4]. Using AlphaFold2 they generated a model of MAGE-A4 bound to a part of RAD18 (Figure). Experimental methods such as nuclear magnetic resonance (NMR), mutagenesis, and the EEF and biophysics platforms of the PSB were used to validate the Alphafold2 model. Further biochemical and cellular experiments showed that the binding of MAGE-A4 specifically hampers the self-ubiquitination activity of RAD18 that in turn leads to its higher stability in cells. Specific mutations in MAGE-A4 result in the loss of MAGE-A4’s ability to stabilize RAD18 in cells. Importantly, the group discovered that most human MAGE proteins possess a ligase-binding cleft (LBC) that is responsible for binding to ubiquitin ligases. In addition, researchers have also unveiled a novel intramolecular interaction in RAD18 which is essential for its ubiquitination activity towards PCNA. Overall, these findings, in particular the LBC of MAGE proteins, will be of particular interest to researchers as they target these molecules for therapeutic purposes.
S. Bhogaraju (EMBL)
[1] Gao Y, Mutter-Rottmayer E, Greenwalt AM, Goldfarb D et al (2016) Nat. Commun., 7, 12105
[2] Li X, Zou S, Zhou L, Gao A et al (2022) Cancer Med., 11, 3809-3819
[3] Weon JL and Potts PR (2015) Curr. Opin. Cell Biol., 37, 1-8
[4] Griffith-Jones S, Álvarez L, Mukhopadhyay U, Gharbi S et al. (2024) EMBO J., 43, 1273-1300
Figure: AlphaFold2 predicted model of MAGE-A4 bound to a peptide of RAD18. MAGE-A4 is depicted in surface representation with conservation of residues among MAGE proteins mapped onto the surface.
