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.