Fig 2: UBR5 binds to mitotic checkpoint proteins and promotes their ubiquitylation. (A) Binding of mitotic checkpoint proteins to UBR5 in extracts from mitotic and asynchronous HeLa cells. Extracts from nocodazole-arrested (“mitotic”) or logarithmically growing (“asynchronous”) HeLa cells were prepared as described previously (7). Immunoprecipitation (IP) of extracts with an anti-UBR5 antibody or with nonimmune rabbit IgG was carried out as described in SI Appendix, SI Materials and Methods. Results are expressed as the percentage of immunoprecipitated proteins relative to the corresponding inputs. Inputs show 10% of the amounts of the indicated proteins in mitotic or asynchronous extracts used for immunoprecipitation. Numbers on the Right indicate the electrophoretic migration of marker proteins (in kDa). (B) Binding of UBR5 to individual recombinant mitotic checkpoint proteins. The indicated recombinant mitotic checkpoint proteins (100 nM) were incubated with recombinant his6-UBR5 (at a quantity similar to that in 40 μg protein of mitotic HeLa cell extract) in a buffer containing: 50 mM Tris⋅HCl (pH 7.2), 20% (vol/vol) glycerol, 1 mg/mL bovine serum albumin (BSA), and 1 mM dithiothreitol (DTT). Following incubation for 1 h at 23 °C, the samples were immunoprecipitated with anti-UBR5 polyclonal antibody or with nonimmune rabbit IgG. Immunoprecipitated material was resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for the indicated proteins. Results are expressed as the percentage of immunoprecipitated material relative to input. Numbers on the Right (kDa) indicate the electrophoretic migration position of marker proteins. (C) Ubiquitylation of individual mitotic checkpoint proteins by recombinant UBR5. The indicated purified recombinant checkpoint proteins (100 nM, each) were incubated in a volume of 30 μL with a ubiquitylation mixture consisting of 40 mM Tris⋅HCl (pH 7.6), 5 mM MgCl2, 1 mg/mL BSA, 1 mM DTT, 150 nM E1 (Enzo BML-UW9410), 600 nM UbcH5ɑ/UBE2D1 (Boston Biochem E2-616), 100 μM ubiquitin, and 2 mM adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP), in the presence or absence of recombinant his6-UBR5 (supplemented at a quantity similar to its amount in 40 μg protein of HeLa cell extract). Following incubation at 37 °C for 1 h, samples were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Numbers on the Right indicate the electrophoretic migration position of marker proteins.

Fig 3: DTX E3 ligases ubiquitylate an ADP-ribosylated peptide and protein(A) DTX2-catalysed ubiquitylation of an ADP-ribosylated histone H3-derived peptide results in a peptide carrying a composite ADPr-Ub modification.(B) A schematic showing the cleavage sites of ARH3 and SARS-CoV2-PLpro within peptide-ADPr-Ub.(C) The conjugate between Ub and an ADP-ribosylated peptide is obtained by incubation of histone H3-derived biotinylated peptide-ADPr with DTX2 RING-DTC (residues 390-622), Ub, E1, E2 and ATP. The same reaction with an unmodified biotinylated H3 peptide was performed as a control. The products were then incubated with indicated hydrolases, revealing sensitivity of the peptide-ADPr-Ub adduct to ARH3, consistent with ubiquitylation of peptide-ADPr on the ADPr moiety as illustrated in B. The samples were analysed by SDS-PAGE, and either the gel was stained with Commassie (left) or proteins were transferred onto a membrane and immunoblotted with anti-Ub (middle) or anti-biotin (right, detecting biotinylated peptides) antibodies. The arrows indicate ARH3 and PLpro while the asterisk represents a contaminant present in peptide-ADPr.(D) An assay analogous to that in C probing the dependence of peptide-ADPr-Ub formation on Asp117 of E2. DTX2-catalysed ubiquitylation of an ADP-ribosylated peptide in the presence of E1, E2 and ATP does not require Asp117 of the E2 UBCH5A, consistent with the ubiquitylation of the ADPr moiety.(E) PARP10 was pre-incubated with NAD+ or buffer and ubiquitylated with a mixture of unlabelled and biotinylated Ub by DTX2 RING-DTC (residues 390-622) in the presence of E1, E2 and ATP. Note that SUMO-tagged E2 was used. The results were visualised with a Coomassie stain (left) and anti-biotin antibody (right, detecting biotin-Ub). DTX2 ubiquitylates auto(ADP-ribosyl)ated PARP10 but not its unmodified form. The dispensability of Asp117 of E2 for the reaction suggests Ub attachment via ADPr.

Fig 4: Differences between WWE domain-containing and KH domain-containing DELTEX E3s.(A) Domain organisation of DELTEX family E3s. DTX1, DTX2 and DTX4 are classified into WWE domain-containing DELTEXes; DTX3 and DTX3L are classified into KH domain-containing DELTEXes. (B) Chemical structure of poly(ADP-ribose) chain, PAR. (C) DTX2-RD and DTX3L-RD ubiquitylate PAR. The arrows indicate PARG. (D) DTX3-RD and DTX3L-RD, but not DTX1-RD, DTX2-RD as well as DTX3L-RD, are able to ubiquitylate nucleic acids. E21_DNA_A was incubated with E1, E2 UbcH5A, ATP, Ub and individual DELTEX E3s’ RD domain, then the reactions were analysed on an SDS-PAGE gel and Urea gel and visualized using the Molecular Imager PharosFX system (BioRad). Each experiment has been completed in triplicate.

Fig 5: Biochemical characterization of DTX3L-RD-catalysed nucleic acids ubiquitylation.(A) Structural analysis showing that the shared AMP moiety in NAD+ and ADPr inserts deeply in the binding pockets of the DTC domains from DTX1 or DTX2. The AMP moiety is indicated by black dashed box. (B) Chemical structures of the E21_DNA_A and E21_RNA_A used in this study. (C) Biochemical reconstitution of E21_DNA_A ubiquitylation. E21_DNA_A-Ub was obtained by incubation of DTX3L-RD and E1, E2 UbcH5A, ATP and Ub. Omitting any of these components blocked the E21_DNA_A ubiquitylation. The reactions were divided into two parts. One part was analysed on an SDS-PAGE gel and visualized by first Molecular Imager PharosFX system (BioRad) and then Coomassie staining. Another part was loaded on a pre-run 20% denaturing urea PAGE gel. The gels were run at 6 W/gel and following visualization using the Molecular Imager PharosFX system (BioRad). (D) As in (B), E21_RNA_A was used as substrate for the ubiquitylation reactions. Each experiment has been completed in triplicate.