DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids

Abstract

Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3'-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3' overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.

Keywords: DTX3L; biochemistry; chemical biology; human; nucleic aicds; ubiquitin; ubiquitin ligase.

Figure 1.. DTX3L catalyses the 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 constructs is shown above the model. (B) DTX3L KHL2 (232-304) prediction shown in green overlaid with the third K Homology (KH) domain of KSRP in complex with AGGGU RNA sequence (PDB 4B8T) shown in brown. (C) DTX3L KHL5 (448-511) prediction shown in green overlaid with the MEX-3C KH1 domain in complex with GUUUAG RNA sequence (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. Data points for (D) and (E) are shown in Figure 1—source data 1 and 2, respectively. (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, and 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, and Mg²⁺-ATP. Asterisks in (F) and (H) indicate contaminant bands from single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Raw unedited and uncropped gel images for (F–L) are shown in Figure 1—source data 3 and 4, respectively.

Figure 1—figure supplement 1.. DTX3L binds and ubiquitinates single-stranded nucleic acids (ssNAs).

(A) Schematic of the 5’ 6-FAM modification. (B) Fold change of fluorescence polarisation of 6-FAM-labelled single-stranded DNA (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 single-stranded RNA (ssRNA) R1-9. Data points for (B–E) are shown in Figure 1—figure supplement 1—source data 1–4, respectively. (F) Schematic of RNA, DNA, and ADPr. (G) Fluorescently detected SDS-PAGE gel of in vitro autoubiquitination of DTX3L FL and 232-C in the presence of E1, UBE2D2, fluorescent-labelled Ub and Mg²⁺-ATP. (H) Coomassie-stained SDS-PAGE gel of lysine discharge reactions showing the disappearance of UBE2D2~Ub over time in the presence of FL, 232-C or absence of E3. (I) 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 Figure 1H). Arrows indicate potential ~15 kDa Ub-DNA band. (J) Schematic of IRDye 800 modification. (K) 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. (L) Schematic of the 3’ 6-FAM modification. Asterisks in (K) indicate contaminant band from ssDNA. Raw unedited and uncropped gel images of (G–I), (K) are shown in Figure 1—figure supplement 1—source data 5 and 6, respectively.

Figure 2.. Ubiquitin (Ub) modification of single-stranded DNA (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 single-stranded RNA (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, Poly(ADP-ribose) glycohydrolase (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 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 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 bands from ssDNA or ssRNA. Raw unedited and uncropped gel images are shown in Figure 2—source data 1 and 2, respectively.

Figure 3.. Nucleotide sequence requirements for ubiquitin (Ub)-DNA formation.

Fluorescently detected SDS-PAGE gel of in vitro ubiquitination (in the presence of E1, UBE2D2, Ub, and Mg²⁺-ATP) of (A) 6-FAM-labelled single-stranded DNA (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 bands from ssDNA. Raw unedited and uncropped gel images are shown in Figure 3—source data 1 and 2, respectively.

Figure 4.. DTX3L DTC domain binds and facilitates ubiquitin (Ub)-DNA formation.

(A) ¹H-¹⁵N heteronuclear single-quantum coherence (HSQC) spectra of ¹⁵N-DTX3L-RD (black), ADPr-¹⁵N-DTX3L-RD (orange), and single-stranded DNA (ssDNA) D30-¹⁵N-DTX3L-RD (blue). Red arrows indicate cross peaks that shift upon titrating with adenosine 5′-diphosphate (ADP)–ribose (ADPr) or ssDNA. (B) Close-up view of the cross peak indicated by the black box in (A) upon titration of specified molar ratios of ADPr with ¹⁵N-DTX3L-RD. (C) Close-up view of the cross peak indicated by the 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) Western blot of in vitro ubiquitination of biotin-NAD⁺ by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg²⁺-ATP and treated with excess ssDNA D31. (F) Kinetics of Ub-D4 and Ub-F-NAD⁺ formation catalysed by DTX3L-RD. Data from two independent experiments (n=2) were fitted with the Michaelis–Menten equation and k_cat/K_m value for D4 (5457  M^–1 min^–1) was calculated. k_cat/K_m value for F-NAD⁺ (1190  M^–1 min^–1) was estimated from the slope of the linear portion of the curve. (G) 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. (H) Structure of DTX3L-DTC domain (cyan; PDB: 3PG6). The sidechains of H707, Y719, and E733 are shown in sticks. (I) 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 (I) indicate contaminant band from ssDNA. Raw unedited and uncropped gel images of (D), (E) and (I) are shown in Figure 4—source data 1 and 2, respectively. Data points for (F) are shown in Figure 4—source data 3.

Figure 4—figure supplement 1.. DTX3L-RD binds adenosine 5′-diphosphate (ADP)–ribose (ADPr) and single-stranded nucleic acids (ssNA).

(A) ¹H-¹⁵N heteronuclear single-quantum coherence (HSQC) spectra of ¹⁵N-DTX3L-RD (black) and after the addition of ADPr (orange) (related to Figure 4A). (B) ¹H-¹⁵N HSQC spectra of ¹⁵N-DTX3L-RD (black) and after the addition of single-stranded DNA (ssDNA) D30 (blue) (related to Figure 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 increasing concentrations of 6-FAM-labelled ssDNA D4 by DTX3L-RD in the presence of E1, UBE2D2, Ub, and Mg²⁺-ATP. (E) Replicate of (D). (F) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of increasing concentrations of F-NAD⁺ by DTX3L-RD in the presence of E1, UBE2D2, Ub, Mg²⁺-ATP. A known volume of 100 µM F-NAD⁺ was pipetted onto Whatman filter paper and scanned alongside the gel for quantification. (G) Replicate of (F). (H) 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, and Mg²⁺-ATP. Asterisks in (D) and (E) indicate contaminant bands from ssDNA. Raw unedited and uncropped gel images are shown in Figure 4—figure supplement 1—source data 1 and 2, respectively.

Figure 5.. 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, DTX3L^R-DTX2^D, or DTX2^R-DTX3L^D and separated by SDS-PAGE. (H) Fluorescently detected SDS-PAGE gel of in vitro ubiquitination of 6-FAM-labelled ssDNA D4 by DTX3L-RD, DTX3L^R-DTX2^D, or DTX2^R-DTX3L^D in the presence of E1, UBE2D2, Ub, Mg²⁺-ATP. (I) Schematic diagrams showing the proposed mechanism of ubiquitination of substrates by DTX3L-RD. Asterisks in (A–F) and (H) indicate contaminant bands from ssDNA. Raw unedited and uncropped gel images of (A–H) are shown in Figure 5—source data 1 and 2, respectively.

Figure 5—figure supplement 1.. Properties of DELTEX (DTX) family DELTEX C-terminal (DTC) domains.

(A) Sequence alignment of human DTX family RING-DTC domains. Starting and ending residues of RING-DTC domains are indicated. Conserved residues are highlighted in yellow. A black arrow indicates residue that stacks with adenosine 5′-diphosphate (ADP)–ribose (ADPr), red arrows indicate conserved catalytic residues, and green arrows indicate AR motif present in DTX1, 2, and 4. Insertions are indicated. (B) Structure of DTX2 RING domain (from PDB: 6Y3J). The insertions and N-terminal helix are colored in red and the conserved RING domain region is colored yellow. (C) Alphafold2 model of DTX3L RING domain (light blue) displayed in the same orientation as in B. (D) Top: cartoon representation of the structure of DTX2 DTC domain (green; PDB: 6Y3J). Sidechains of the AR motif are shown as sticks. The DTC pocket that binds ADPr and the bulged AR motif are indicated by arrows. Bottom: as in the top panel but with surface representation. (E) Top: cartoon representation of the structure of DTX3L DTC domain (light blue; PDB: 3PG6). Bottom: as in the top panel but with surface representation. The absence of an AR motif in DTX3L causes a slight structural change near the ADPr-binding pocket, leading to the loss of the bulged loop, which results in a slightly extended groove.

Author response image 1.. Fold change of fluorescence polarisation of 6-FAM-labelled ssDNA D4 upon titrating with DTX3L variants.

DTX3L KH domain fragments were expressed with a N-terminal His-MBP tag to increase the molecular weight to enhance the signal.

Author response image 2.. Fluorescently detected SDS-PAGE gel of in vitro ubiquitination catalysed by DTX3L-RD in the presence ubiquitination components and 6-FAM-labelled ssDNA D4 or D31.