Fig. 8: REV3 interacts with SHLD3 mediated C-REV7-O-REV7 and locates at DSB sites.

a ITC measurement of interaction between REV7^E35A-SHLD3(1–82)-REV7^K129A and REV3(1847-1906). REV7^K129A was first saturated by excessive REV7^E35A-SHLD3(1–82) as shown in Fig. 2d. The calculated N and K_D are indicated as described in Fig. 2d. b Gel filtration profiles show the interaction between MBP-REV3(1847-1906) and REV7^E35A-SHLD3(1–82)-MBP-REV7^K129A (short as 1906, E35A-1–82 and K129A respectively in the figure). MBP-REV7^K129A was first saturated by excessive REV7^E35A-SHLD3(1–82) as shown in red line. Co-elutions were analyzed by SDS-PAGE and stained by Coomassie brilliant blue. c ITC measurement of interaction between REV7^E35A-SHLD3(1–82) and REV7^K129A-REV3(1847-1906). REV7^K129A was first saturated by excessive REV3(1847-1906). The calculated N and K_D are indicated as described in Fig. 2d. d Gel filtration profiles show the interaction between MBP-REV3(1847–2021) and REV7^WT-SHLD3(1–82) (short as MBP-1847–2021 and WT-1–82 in the figure). Co-elutions were analyzed by SDS-PAGE and stained by Coomassie brilliant blue. e S-tag-HA-SHLD3 was co-expressed with GFP-tagged REV3(1847–2021) (short as GFP-REV3-2021 in the figure) in HEK293T cells. Indicated cells were treated with DMSO or 1 μM doxorubicin for 24 h. S-tag-HA-SHLD3 and its associated proteins were purified by S-protein beads. HA and GFP antibodies were used to detect S-tag-HA-SHLD3 and GFP-tagged REV3(1847–2021), respectively. The input panel shows the transfection efficiency and the S-tag pulldown panel shows the interaction between SHLD3 and REV3(1847–2021). Source data are provided as a Source Data file. f Schematic representation showing conserved domains of human REV3 and the truncation used in laser micro-irradiation assay. The N-terminal domain (NTD) and polymerase domain are shaded in yellow green, REV7 binding motif (RBM) in yellow, and the positively charged domain (PCD) in blue. Domain boundaries are indicated by residue numbers. In the REV3 deletion construct REV3^TR1, predicted unstructured portions with boundaries marked by residue numbers were deleted. FL: full length. g, h mCherry-SHLD3 and GFP-REV3 localization to DNA damage were monitored after laser micro-irradiation of 293FT and HeLa cells. Cells were imaged 5 min after damage induction except REV3^FL (10 min) due to its large size. n = 2 biologically independent experiments in b, d, e and h, except g was assessed once.