Figure 2.

hTRAF6 interacts with hDNA2 and catalyzes its ubiquitination. (A) 293T cells were transfected with a 3xFlag-hDNA2 or an empty control vector (EV). The 3xFlag-hDNA2 complex was pulled down using anti-Flag M2 beads. The endogenous hTRAF6 in complex with 3xFlag-hDNA2 was detected using anti-hTRAF6 antibody. (B) 3xFlag-hDNA2 and myc-hTRAF6 were co-expressed in 293T cells. In control reactions, myc-hTRAF6 was co-expressed with 3xFlag-GFP and 3xFlag-hDNA2 was co-expressed with a non-specific peptide encoded by the empty myc vector. 3xFlag-hDNA2 and the myc-hTRAF6 complex were pulled down using anti-Flag M2 or anti-myc affinity beads. myc-hTRAF6 and 3xFlag-hDNA2 were detected by western blot analysis using anti-myc or anti-Flag antibodies. (C) 293T cells co-expressing 3xFlag-hDNA2 and myc-hTRAF6 were treated with DMSO or CPT (1 μM, 4 h). 3xFlag-hDNA2 and myc-hTRAF6 were pulled down and analyzed by western blot, as described for panel (B). (D) In vitro reconstitution of hDNA2 ubiquitination using ubiquitin (Ubn) and purified recombinant myc-hTRAF6, 3xFlag-hDNA2, ubiquitination enzymes E1 and E2, and mixed E3 ligases. (E) 3xFlag-hDNA2 was co-expressed with 6His-tagged Ubn (6His-Ubn) and myc-hTRAF6 in 293T cells. Total hDNA2 was isolated using anti-Flag M2 beads and the ubiquitination status of 3xFlag-hDNA2 was analyzed by western blot analysis using an anti-6His antibody. (F) 3xFlag-hDNA2 was co-expressed with 6His-Ubn and WT myc-hTRAF6 or C70A (ubiquitin E3 ligase activity-deficient) mutant myc-hTRAF6 in 293T cells. Total ubiquitinated proteins were isolated using Ni-NTA beads under denaturing conditions (8 M urea). 6His-Ubn-linked hDNA2 was detected by western blot analysis using an anti-Flag antibody. (G) 3xFlag-hDNA2 was co-expressed with HA-tagged WT, K27R, K48R or K63R ubiquitin (HA-Ubn) and myc-hTRAF6 in 293T cells. Total hDNA2 was isolated using anti-Flag M2 beads, and the ubiquitination status of 3xFlag-hDNA2 was analyzed by western blot analysis using an anti-HA antibody.