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. 2021 Mar 11;12:1591. doi: 10.1038/s41467-021-21850-9

Fig. 2. Repair of lesions on HBV rcDNA plus- and minus-strands with purified human factors.

Fig. 2

a Schematic of examining the kinetics of repair on plus- and minus-strands simultaneously by Southern blot. One half of the repair products were used to monitor cccDNA formation by agarose gel electrophoresis; the other half was digested to generate four fragments of interest: Pa and Pb containing lesions on the plus-strand; Ma and Mb harboring lesions on the minus-strand. These four fragments of repair intermediates (denoted by “?”) could be detected by Southern blot as illustrated by idealized blots. The location of the repair intermediates are arbitrarily chosen and indicated by “?”. Green line, biotinylated flap; B, biotin; red line, RNA primer. b Schematic depicting the four fragments in digested unrepaired RrcDNA. c A time course cccDNA formation assay with NeutrAvidin-RrcDNA (NA-RrcDNA, lanes 1–7) or RrcDNA (lanes 8–14) and five purified factors, as depicted in a. “% cccDNA formed” was calculated as described in Fig. 1e, f. d The repair of the plus-strand Pa fragment is monitored by Southern blot. “*” indicates extension products of Pa that reach the 5′ terminus of Pb. e The repair of plus-strand RNA primer-containing Pb fragment is monitored by Southern blot. Traces (plotted with gel-profile function in ImageJ) denote unprocessed (blue arrow head) and processed product (orange arrow head) of Pb fragments, respectively, at 1 min. f–g Repair of minus-strand Ma and Mb fragments are monitored by Southern blot. h Removal of biotin-containing flap in Ma is detected by Streptavidin blot. i The repair efficiency of plus- and minus-strands is calculated from e and f and plotted. “% repaired” is calculated by dividing the band intensities of fully repaired Pa or Ma by the sum of the band intensities of unrepaired, intermediate, and fully repaired Pa or Ma. M, marker; Rrc, RrcDNA; rL, recombinant linear RrcDNA; ccc, cccDNA. All experiments were repeated three times. The lines connect the average value of three measurements at each time point. P values are 0.00001, 0.000002, 0.000003, 0.000002, 0.00002, and 0.00005 at time points between 1 and 60 min. Source data are provided as a Source Data file.