CK2α O-GlcNAcylation affects its conformational stability and modulates its ability to phosphorylate RAD51

Homologous recombination (HR) is a DNA repair pathway which contributes to maintaining genomic integrity. This pathway, which is often dysregulated in cancer treatments resistance, is dependent on RAD51 protein. RAD51 activity is regulated by post-translational modification, particularly its phosphorylation on threonine 13 by casein kinase 2 sub-unit α (CK2α), which facilitates its recruitment to the DNA damage site. CK2α carries an O-GlcNAc, which is known to affect its ability to phosphorylate certain substrates. Recently, several studies have highlighted a modulatory role of O-GlcNAcylation on HR, although no link to RAD51 has been established. Using co-transformation of CK2α with OGT, we obtained an O-GlcNAcylated CK2α protein in bacterio, that we were able to compare to its unmodified counterpart. Structural analyses using Nano DSF and circular dichroism revealed a local stabilizing effect of O-GlcNAcylation on the regions carrying this glycan. Finally, a kinetic comparison of RAD51 phosphorylation by CK2α or CK2α O-GlcNAcylated revealed that O-GlcNAcylation increase CK2α ability to phosphorylate RAD51 at threonine 13. Overall, this study demonstrates for the first time a potential indirect role of O-GlcNAcylation in modulating RAD51 activity.