Figure 3: Adprt1a is a DNA strand break-induced mono-ART that ADP-ribosylates H2Bv3 at E18 and E19.

(A) Wild-type or catalytic dead (Adprt1a^cd) His-tagged Adprt1a was expressed and purified from bacteria (left) and employed in ADP-ribosylation assays using ³²P-labeled NAD⁺ in the absence or presence of sheared salmon sperm DNA (sssDNA). Following SDS-PAGE, ADP-ribosylated proteins were detected by auto-radiography. (B) ADP-ribosylation assays were performed using His-Adprt1a as in (A) with the exception that pUC19 DNA either uncut, or cut with enzymes to produce the indicated numbers of breaks, was employed in assays instead of sssDNA. (C) His-Adprt1a (right) and human PARP1 (left) were auto-ribosylated by incubation with biotin-NAD⁺ prior to incubation with increasing concentrations of MacroD1 or PARG as indicated. ADP-ribosylated proteins were detected by Western blotting with Streptavidin conjugated HRP. (D) His-H2Bv3 or His-H2Bv3 lacking the N-terminal 25 amino acids (H2Bv3Δ25) were expressed and purified from bacteria (upper panel). ADP-ribosylation reactions were performed with Adprt1a in the presence of sssDNA as indicated. ADP-ribosylated proteins were detected as in (C). (E) Sequence alignment of the N-terminal 24 amino acids of Dictyostelium H2Bv3 with the equivalent regions of H2B from a variety of species. Identical amino acids are highlighted and potential ADP-ribosylation sites in the Dictyostelium protein indicated (*). (F) Wild-type His-H2Bv3, or His-H2Bv3 mutated at E18, E19, or both E18 and E19 were expressed and purified from bacteria (upper panel). Proteins were employed in ADP-ribosylation reactions as in (D).