HBx impedes the DNA repair of UV damaged plasmid DNA in-vitro. (A) In vitro repair of UV-damaged pBR322 DNA using yeast lysates expressing HBx and its mutants. The repair reaction contained, 0.3 μg un-irradiated pUC18 and 0.3 μg UV-irradiated pBR322 substrate, was performed as discussed in the experimental procedure. Control plasmid (lane 1); HBx expressing plasmid (lane 2); and its mutant Glu120 (lane 3); Glu 121 (lane 4); Glu 124 (lane 5) and Glu 125 (lane6). Reactions were incubated for 6 hours at 30°C. Reactions were stopped by the addition of EDTA and then incubated with RNAse, SDS and proteinase K. Plasmids were digested with HindIII and loaded on 1% agarose gel. After overnight electrophoresis, the gel was photographed under near-UV transillumination with Polaroid film (right panel) and an autoradiograph of the dried gel was obtained (left panel) (B) HBx is unable to repair the damaged plasmid DNA in Rad1 and Rad51 mutant yeast strain. Plasmid p-GAL4 and pGAL4-X were transformed into yeast strains with normal RAD1 and RAD51 genes (lane 1, 2), with deletion of Rad1 (lane 3, 4) and with deletion of RAd51 (lane 5-6). Nuclear extract were assayed for DNA repair of UV-damaged pUC18 DNA (C) HBx is unable to repair damaged plasmid DNA in SSL2 mutant (dead) and temperature sensitive yeast strain. Plasmid p-Gal4 and pGAL4-X were transformed into yeast strains with normal SSL2 (lane 1, 2) mutant SSL2-dead strain (lane 3, 4) and temperature strain (lane 5-6). Nuclear extracts were assayed for DNA repair of UV-damaged pBR322 DNA The yeast ts strain was grown at room temperature (20-21°C).