Specificity and recognition of the ADP-ribosyl-ubiquitin modification in the DNA damage response

Abstract

ADP-ribosylation (ADPr) is a post-translational modification that has regulatory roles in multiple cellular pathways including the DNA damage response and in innate immunity. Recently, it has been uncovered that ADP-ribose can be further modified by a family of ubiquitin E3 ligases, the DELTEXES, which catalyze ubiquitin transfer directly onto ADP-ribose, creating a hybrid ADPr-Ub modification which can be recognized by proteins with dedicated ADPr-Ub binding domains. With this hybrid modification recently been identified in cellular systems, we use a series of in vitro and cellular assays in human cells to investigate the amino acid preference for ADPr-Ub production as well as conditions required for reversal of the modification. We show that ADPr on both serine and glutamate-linked peptides can be ubiquitinated by the RING-DTC domains of DTX2 and DTX3L in vitro and that this can be recognized by RNF114, RNF138 and RNF166 for ubiquitin chain elongation. Finally, we demonstrate that DTX2 rather than DTX3L plays a role in ADPr-Ub production at sites of DNA damage to promote the recruitment of RNF114, RNF138, and RNF166 in an HPF1-independent manner.