Figures and data
DTX3L catalyses ubiquitination of single-stranded nucleic acids.
(A) Cartoon representation of the AlphaFold model of DTX3L. Domains are coloured according to model confidence. Domain architecture of DTX3L is shown above the model. (B) DTX3L KHL2 (232–304) prediction shown in green overlaid with the third KH domain of KSRP (PDB 4B8T) shown in brown. (C) DTX3L KHL5 (448–511) prediction shown in green overlaid with the MEX-3C KH1 domain (PDB 5WWW) shown in purple. (D) Fold change of fluorescence polarisation of 6-FAM-labelled ssDNA D1-9 upon titrating with full-length DTX3L. (E) As in (D) but with 6-FAM-labelled ssRNA R1-9. (F) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D1-9 oligonucleotides by FL DTX3L in the presence of E1, UBE2D2, Ub, Mg2+-ATP. (G) As in (F) but with 6-FAM-labelled ssRNA R1-9 oligonucleotides. (H) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L variants (FL, full length; RD, RING-DTC domains; R, RING domain; D, DTC domain) in the presence of E1, UBE2D2, Ub, Mg2+-ATP. (I) As in (H) but with 6-FAM-labelled ssRNA R4. (J) Coomassie stained SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg2+-ATP. (K) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 5’ IRDye® 800 ssDNA D10 by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg2+-ATP. (L) Coomassie stained SDS-PAGE gel of in vitro ubiquitination of ssDNA D11 by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg2+-ATP. Asterisks in (F), (H), and (I) indicate contaminant bands from ssDNA or ssRNA. Uncropped gel images for (F–L) are shown in fig. S4.
List of nucleotide sequences used in this study
Ub modification of ssDNA occurs at the 3’ hydroxyl at the 3’ end.
(A) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of ssDNA D4 in which E1, UBE2D2, DTX3L-RD, Ub or Mg2+-ATP have been omitted. (B) As in (A) but with 6-FAM-labelled ssRNA R4. (C) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg2+-ATP subsequently treated with USP2, Benzonase, PARG or not treated (None). (D) As in (C) but with 6-FAM-labelled ssRNA R4. (E) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 and dsDNA D12 oligonucleotides by DTX3L-RD (left panel) and at increased exposure (right panel) in the presence of E1, UBE2D2, Ub, Mg2+-ATP. (F) As in (E) but with 6-FAM-labelled ssDNA D4 (5’ label) and D13 (3’ label) oligonucleotides. (G) Fluorescently detected SDS-PAGE gel of in vitro ubiquitinated of 6-FAM-labelled ssDNA D4 subsequently treated with pH 9.5 buffer for the times indicated. (H) Fluorescently detected SDS-PAGE gel of in vitro ubiquitinated of 6-FAM-labelled ssDNA D4 subsequently treated with 1.5 M NH2OH at pH 9 for the times indicated. (I) As in (E) but with 5’ IRDye® 800 ssDNA D10, D14 and D15 oligonucleotides. Asterisks in (A–C) and (E–H) indicate contaminant band from ssDNA or ssRNA. Uncropped gel images are shown in fig. S4.
Nucleotide sequence requirements for Ub-DNA formation.
(A) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination (in the presence of E1, UBE2D2, Ub, and Mg2+-ATP) of 6-FAM-labelled ssDNA D4, D16, D17 and D18 by DTX3L-RD. (B) 6-FAM-labelled ssDNA D4, D19, D20 and D21 by DTX3L-RD. (C) 6-FAM-labelled ssDNA D4, D22 and D23 by DTX3L-RD. (D) 6-FAM-labelled ssDNA D4, D24 and D25 by DTX3L-RD. (E) 6-FAM-labelled ssDNA D26, dsDNA D27, D28 and D29 by DTX3L-RD. Asterisks indicate contaminant band from ssDNA. Uncropped gel images are shown in fig. S4.
DTX3L DTC domain binds and facilitates Ub-DNA formation.
(A) 1H-15N HSQC spectra of 15N-DTX3L-RD (black), ADPr-15N-DTX3L-RD (orange), and ssDNA D30-15N-DTX3L-RD (blue). Red and black arrows indicate cross peaks that shift upon titrating with ADPr or ssDNA. (B) Close-up view of the cross peak indicated by black arrow in (A) upon titration of specified molar ratios of ADPr with 15N-DTX3L-RD. (C) Close-up view of the cross peak indicated by black arrow in (A) upon titration of specified molar ratios of ssDNA D30 with 15N-DTX3L-RD. (D) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg2+-ATP and treated with excess ADPr. (E) Structure of DTX2-DTC domain (green) bound to ADPr (yellow) (PDB: 6Y3J). The sidechains of H582, H594 and E608 are shown in sticks. Hydrogen bonds are indicated by dotted lines. (F) Structure of DTX3L-DTC domain (cyan; PDB: 3PG6). The sidechains of H707, Y719 and E733 are shown in sticks. (G) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by full length DTX3L WT, H707A, Y719A and E733A in the presence of E1, UBE2D2, Ub, Mg2+-ATP. Asterisks in (D) and (G) indicate contaminant band from ssDNA. Uncropped gel images of (D) and (G) are shown in fig. S5.
Select DTX RING-DTC domains catalyse ubiquitination of ssDNA.
(A) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L-RD or DTX1-RD in the presence of E1, UBE2D2, Ub, Mg2+-ATP. (B) As in (A) but with DTX2-RD. (C) As in (A) but with DTX3-RD. (D) As in (A) but with DTX4-RD. (E) As in (A) but with DTX3L-RD or DTX3L RING with increasing concentrations of DTX3L DTC. (F) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D1-9 by DTX2-RD. A reaction with DTX3L-RD and 6-FAM-labelled ssDNA D4 was included as a positive control. (G) Western blot of in vitro ubiquitination of biotin-NAD+ in the presence of E1, UBE2D2, Ub, Mg2+-ATP, NAD+, biotin-NAD+ with either DTX3L-RD, DTX3LR-DTX2D, or DTX2R-DTX3LD and separated by SDS-PAGE. (H) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L-RD, DTX3LR-DTX2D, or DTX2R-DTX3LD in the presence of E1, UBE2D2, Ub, Mg2+-ATP. (I) Schematic diagrams showing the proposed mechanism of ubiquitination of ssNAs by DTX3L-RD. Asterisks in (A–F) and (H) indicate contaminant band from ssDNA. Uncropped gel images of (A–H) are shown in fig. S5.
DTX3L binds and ubiquitinates ssNAs.
(A) Schematic of the 5’ 6-FAM modification. (B) Fold change of fluorescence polarisation of 6-FAM-labelled ssDNA D1-9 oligonucleotides upon titrating with DTX3L 232-C. (C) As in (B) but with 6-FAM-labelled ssRNA R1-9 oligonucleotides. (D) As in (B) but with DTX3L 232-C/PARP9 509-C. (E) As in (D) but with 6-FAM-labelled ssRNA R1-9. (F) Schematic of RNA, DNA and ADPr. (G) Coomassie stained SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L variants in the presence of E1, UBE2D2, Ub, Mg2+-ATP (from Fig. 1H). Arrows indicate potential ∼15 kDa Ub-DNA band. (H) Schematic of IRDye® 800 modification. (I) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D1-9 in the presence of E1, UBE2D2, Ub, Mg2+-ATP by DTX3L-RD. (J) Schematic of the 3’ 6-FAM modification. Asterisks in (I) indicate contaminant band from ssDNA. Uncropped gel images of (I) are shown in fig. S5.
DTX3L-RD binds ADPr and ssNA.
(A) 1H-15N HSQC spectra of 15N-DTX3L-RD (black) and after the addition of ADPr (orange) (related to Fig. 4A). (B) 1H-15N HSQC spectra of 15N-DTX3L-RD (black) and after the addition of ssDNA D29 (blue) (related to Fig. 4A). (C) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssRNA R4 by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg2+-ATP and treated with excess ADPr. (D) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssRNA R4 by FL DTX3L WT, H707A, Y719A and E733A in the presence of E1, UBE2D2, Ub, Mg2+-ATP. Uncropped gel images of (C) and (D) are shown in fig. S5.
Properties of DTX family DTC domains.
(A) Sequence alignment of human DTX family DTC domains. Starting and ending residues of DTC domains are indicated. Conserved residues are highlighted in yellow. A black arrow indicates residue that stacks with ADPr, red arrows indicate conserved catalytic residues, and green arrows indicate AR motif present in DTX1, 2, and 4. (B) Top: cartoon representation of structure of DTX2 DTC domain (green) bound to ADPr (PDB: 6Y3J). Sidechains of AR motif are shown as sticks. Bottom: as in top panel but with surface representation. (C) Top: cartoon representation of structure of DTX3L DTC domain (blue; PDB: 3PG6). Bottom: as in top panel but with surface representation. (D) Top: Cartoon representation of overlaid structures of DTX2 DTC domain and DTX3L DTC domain. The absence of an AR motif in DTX3L causes a slight structural change near the ADPr-binding pocket. Bottom: as in top panel but with surface representation.
