cerevisiae wild type strain 334 is MATα pep4-3 prb1-1122

ura3-52 leu2-3, 112 regI-50 gal1. Two NER defective yeast strains rad 1 and rad51 were employed in this study. The genotype of Rad1 is (α rad1-2 his3Δ1 leu2-3-112 lys 1-1 trp1-289 ura3-52) and rad 51 (α rad51-1 his3Δ1 leu2-3-112 lys 1-1 trp1-289 ura3-52). Plasmids pUC18 and pBR322 were used for repair synthesis assays and were purified as described [47]. Plasmid pSBDR contains sequences encoded by an HP1 to Taq1 fragment derived from HBV adw strain which includes enhancer 1 element followed by X promoter, the HBx coding sequences and the polyA addition site. In addition, pSBDR contains neomycin resistance marker for selection in eukaryotic cells. UV survival profile of HBx expressing yeast cells Yeast cultures of strain Selleckchem PR171 AZD9668 in vivo 334 containing plasmids, pYES and pYES-Xwt and pYES-Xmutant (as indicated) were grown in 2 ml of YMIN media (0.17% yeast nitrogen base, 1% succinic acid, 0.6% NaOH and 0.5% Ammonium sulfate)

with 2% glucose. Saturated yeast cultures were washed in water and resuspended into 2 ml of sterile water. Then 200 μl of washed cells were added into 2 ml of fresh YMIN media containing 2% glycerol, 2% ethanol and 2% galactose for the induction of HBx and grown with shaking (200 rpm) for 24 h. Various cell dilutions were plated simultaneously onto two sets of YMIN plates containing 2% glycerol, 2% ethanol and 2% galactose. One set of plates was immediately irradiated under a germicidal lamp for various dosages of UV light and another set of control plates was not exposed to UV-irradiation. Plates were then incubated ADP ribosylation factor in dark for

at least 24 h and shifted to 30°C. Colonies were counted to determine the survival fraction. UV survival profile of HBx expressing human liver cells HBx expression plasmid pSBDR and UV-damaged pRC/CMV were co transfected into Chang liver cells. Plates were incubated in dark for 2 weeks in the presence of 0.4 mg/ml of G-418. The number of G-418 resistant clones per 105 cells is plotted. Live cells were counted by staining with trypan blue after transfection and prior to G-418 selection. Yeast nuclear extracts Yeast cells were grown at 30°C in 1 liter YPD medium (1% yeast extracts, 2% Bactopeptone, 2% Dextrose) to logarithmic phase. Cells were harvested by centrifugation for 10 min, washed in water, and suspended at 0.1 g/ml in 0.1 M EDTA pH 8.0/10 mM dithiothreitol. After incubation at 30°C with shaking (50 rpm) for 10 min, cells were pelleted by centrifugation as described above and suspended at 1 ml in YPS solution (1% yeast extract, 2% Bactopepetone and 1 M sorbitol) and yeast lytic enzyme (ICN) was added at 150 U/g of cells. Following incubation at 30°C with shaking (50 rpm) for 2 hrs, ice cold YPS solution was added (10 mg/g of cells). Spheroblasts were pelleted by centrifugation as above and washed three times in the same buffer. Phenylmethanesulfonyl flouride was added (0.