Fig. 7: DExH-box helicase mediated the formation of eccDNAfib-L.
From: DNA repair pathway-related proteins are involved in the circularization step of microDNA eccDNAfib-L
A A schematic diagram of different domains in Polθ and their prokaryotic expression vectors. SDS-PAGE (left) and Western blot (right) for recombinant DExH-box (B), Ski2 family (C), HTH (D), HLD (E), and polymeras (F), respectively. The primary antibodie was anti-6×His tag mouse polyclonal antibody (diluted at a 1:1000). The HRP-labeled goat anti-mouse IgG was used as the secondary antibody at a 1:5000 dilution. The red arrow represented the target protein. G The GAGT-eccDNAfib-L-GAGT was added to a cell-free reaction system containing the puried DExH-box, Ski2 family, HTH, HLD, and polymeras proteins. PCR and Sanger sequencing were used to detect the junction site of eccDNAfib-L. Control, The GAGT-eccDNAfib-L-GAGT was added to a cell-free reaction system containing the purified protein expressed in the E. coli transformaed with pET28a (+). H 1 μM of ART558 (Polθ inhibitor) and 0.25 μg of GAGT-eccDNAfib-L-GAGT were added to the cell-free reaction system containing the purified DExH-box, and incubation at 16°C overnight, the junction site of eccDNAfib-L was detected by PCR. The cell-free reaction system without ART558 was used as a control.
