RAD18 methylation by the methyltransferase SETD6 attenuates DNA breaks

This study investigated the interaction between the SETD6 lysine methyltransferase and RAD18, a key protein in the DNA damage repair pathway. SETD6 belongs to the SET-domain-containing family of proteins, which are known to catalyze protein methylation, a post-translational modification that plays a critical role in regulating protein function, stability, and interactions. Using protein microarray technology, we identified RAD18 as an interactor and substrate of SETD6. We confirmed this interaction through ELISA and immunoprecipitation assays, demonstrating that SETD6 directly binds and methylates RAD18. Using mass spectrometry and site-directed mutagenesis, we identified that RAD18 undergoes mono-methylation at the K73 and K406 residues. Furthermore, we found that RAD18 methylation affects its nuclear localization. Specifically, SETD6 KO cells exhibited increased nuclear RAD18 levels, suggesting that methylation status influences RAD18's shuttling between the cytoplasm and nucleus. Notably, depletion of SETD6 led to elevated markers of DNA damage (γH2AX) and increased DNA breaks, as evidenced by comet assays. Restoring SETD6 activity significantly reduced DNA damage, while a catalytic inactive mutant did not have this effect, underscoring the importance of SETD6's enzymatic function. Overall, our results demonstrate that SETD6-mediated methylation of RAD18 is essential for attenuating DNA breaks, thereby regulating its cellular localization and function in maintaining genomic integrity.