Fig. 1: K120 is a major SUMO conjugation site of DHX9.
From: DHX9 SUMOylation is required for the suppression of R-loop-associated genome instability
a A primary structure of DHX9, with an SFB-tag at its N-terminus, shows the helicase core and the location of five putative SUMO2 conjugation sites. Colored boxes represent specific features: the DExH-box colored in blue, the NLS in yellow, and boxes in magenta (I and II) for dsRBD1 and dsRBD2, respectively. b Sequences of five putative SUMO2 acceptor sites mapped by system-wide studies. c HeLa cells transfected with SFB-DHX9 (8 μg) alone or with HA-SUMO1 (4 μg) or HA-SUMO2 (3 μg) were used for FLAG affinity purification under denaturing conditions. Protein levels and SUMO conjugation on SFB-DHX9 were determined by Western blot with the indicated antibodies. d A His-GST-tagged DHX9 fragment (1–399) purified from E. coli was subjected to reconstituted reactions to examine DHX9 SUMOylation in vitro. Levels of recombinant DHX91–399 and SUMO-modified DHX91–399 were determined by Western blot using anti-DHX9 antibody. e DHX9 SUMOylation is not regulated by DNA damage. HeLa cells transfected with SFB-DHX9 alone or with HA-SUMO2 were treated with DMSO, HU (4 mM), or CPT (2 μM) for 1 h. SUMOylation of DHX9 and protein levels were determined by Western blot. f, g DHX9 K120 is the major SUMO2 acceptor site. The HA-SUMO2 (f) and endogenous SUMO2/3 (g) conjugation of SFB-DHX9 variants purified by the anti-FLAG magnetic beads was analyzed by Western blot. h Bacterially expressed recombinant DHX91–399 (WT or K120R) were subjected to test DHX9 SUMOylation in vitro. DHX9 SUMOylation was assessed by Western blot. i–k SFB-DHX9WT, but not SFB-DHX9K120R associated SUMO2/3 in the nucleus. HeLa derivative clones transfected with siDHX9 were used for expressing the vector, wild-type (WT), or K120R DHX9 upon doxycycline induction, followed by PLA analysis using the indicated antibodies. i Representative images of PLA analysis. j Quantification of PLA foci-positive cells (red). The black line indicates the median. The number of PLA foci per nucleus was counted from 150 nuclei under each condition and analyzed by a two-sided Mann–Whitney U test. k Levels of SFB-DHX9 in different stable cell lines were determined by Western blot. Source data are provided as a Source Data file.
