This was compared with non-expressing and HBx mutant expressing c

This was compared with non-PRT062607 nmr expressing and HBx mutant expressing cell lysates. Wang and co-workers [47] developed a fairly simple and effective assay to monitor DNA repair in vitro. This assay relies on the repair synthesis of a plasmid which has been previously treated with a base-damaging agent N-acetoxy-2-acetylaminofluorene (AAAF) or UV irradiation. Damaged plasmids are incubated with wild type

yeast cell-free extracts and32P-labeled dCTP. Radioactivity incorporated into the damaged plasmid during DNA repair is observed by agarose gel electrophoresis followed by autoradiography. By employing the mutant alleles of RAD3 and SSL2, Wang and co-workers [47] were able to define a functional role for yeast TFIIH in DNA repair. We employed this assay to determine the effect of HBx on DNA repair process in vitro. To control the specificity of in vitro DNA repair reaction, we also used TFIIH (ssl2) mutant and NER defective rad 1 and rad51 deletion yeast strains as controls. First, UV irradiated plasmid pBR322 was subjected to DNA repair in vitro, with extracts of wild type yeast strain 334 and those transformed with pYES-2

(vector alone), pYES-X (HBx expressing vector) and its mutants Glu 120, see more Glu 121, Glu 124 and Glu 125. Un-irradiated plasmid pUC18 DNA was used as a control. Yeast lysates were prepared 16 hr after treatment with 2% galactose for the expression of HBx and its mutant proteins. HBx and its mutant proteins were expressed equally in these yeast strains

as confirmed by Western blotting (data not shown). Figure 5A shows the results of this experiment. The repair synthesis of UV irradiated plasmid pUC18 using the yeast crude extracts transformed with vector alone (lane 1), HBx expressing vector, (lane 2) and HBx mutants Glu 120 (lane 3), Glu 121 (lane 4), Glu 124 (lane 5) and Glu 125 (lane 6). The incorporation of32P[dCTP] as a measure of DNA repair is shown in Figure 5. These results clearly suggest that HBx expressing yeast lysates are defective in repairing the UV-damaged DNA in vitro (compare lane 1 with lane Bcl-w 2). HBx mutant Asp 113 that has retained the ability to interact with TFIIH (Figure 2A-C) also retains the ability to impede the DNA repair process like wild type HBx (lane 3). Yeast lysates expressing other mutants of HBx showed varying degrees of DNA repair efficiencies (lanes 4-7). More importantly, HBx’s mutant Glu 120 which failed to interact with TFIIH also failed to influence the repair process in vitro (lane 3). The results shown in Figure 5A are encouraging, as no incorporation in the un-damaged pBR322 DNA was observed. To further confirm that non-specific incorporation of radioactivity has not occurred in this reaction, we used HBx expressing NER defective yeast lysates. Two mutant yeast strains with deletions in Rad-1 and Rad-51 were transformed with HBx expressing plasmid pGAL4-X and a control plasmid pGAL4.

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