Both Rad-1 and Rad-51 NER defective lysates showed
no incorporation (lanes 3 and 5). HBx expression in these mutant yeast lysates had no effect on the repair reaction (lane 4 and 6). This suggests that indeed specific DNA repair reaction has occurred in Figure 5A. These results are consistent with the hypothesis that HBx expressing wild type yeast lysates Sotrastaurin have diminished DNA repair efficiency of UV-damaged plasmid DNA. Figure 5 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 Napabucasin ic50 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 why and pGAL4-X were transformed into yeast strains with normal RAD1 and RAD51 genes (lane 1, 2), with GW-572016 molecular weight 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). Next, we examined the ability of HBx to alter DNA excision repair reaction in a TFIIH mutant yeast strain (Figure 5C). Wild type yeast strain and two TFIIH mutant yeast strains ssl2 (dead) and ssl2 (ts) [37] were transformed with a control plasmid pGAL4 and HBx expressing pGAL4-X DNAs. Yeast lysates were prepared as described. UV-damaged pBR322 DNA was used. Consistent with our previous results, HBx expression in wild type strain diminished the ability to repair the DNA (lane 2). TFIIH mutant yeast lysates with HBx (lane 4 and 6) or without HBx (lanes 3 and 5) were equally deficient in DNA repair synthesis, suggesting that HBx impinge its influence on DNA repair via TFIIH. In summary, using myriad experimental strategies, our results implicate HBx in DNA repair process via its physical interactions with the helicase components of TFIIH.