Figure 4

Significance of H-N-H motif in nuclease activity of Usp. (A) Sequence alignment of H-N-H motif domain of wild-type and mutant Usps (amino acid position from 309 to 346). Stars indicate the consensus residues in H-N-H motif[14]. Residues without substitution were indicated with dots. (B) Nuclease activity assay for mutant Usps. Linear pUC 18 (40 ng) was incubated with indicated concentrations of free Usps or Usp/OrfU1-His complexes. The indicated concentration for Usp/OrfU1-His was the concentration of WT or mutant Usps in each complex. After 2 h incubation, DNA degradation was checked by electrophoresis in 1.0% (w/v) agarose gel. (Upper panel). Quantitative analysis was done by measuring the intensity of each band (Lower panel). Data are means ± SD of values from three experiments. (C) Colony formation by E. coli DH5α (left panel) or E. coli BL21(DE3) (right panel) after transformation with plasmids encoding mutant Usp alone. The plasmids encoding wild-type or mutant Usps alone were transformed into E. coli DH5α or E. coli BL21(DE3) and selected on kanamycin containing LB agar plate. The number of colony observed on the Φ100 mm plate by each transformed E. coli after overnight incubation at 37°C is indicated on y-axis. Data are means ± SD of values from three experiments.