FIG. 5.

Nick closure analysis of the effect of Ssh10b on DNA supercoiling. Ssh10b and the single-nick pUC18 plasmid were mixed at various protein/DNA mass ratios: 0, 0.5, 1.0, and 1.5. Following incubation at an assay temperature (25, 45, 60, or 80°C), T4 DNA ligase (for reactions at 25 and 45°C) or Pfu DNA ligase (for reactions at 60 and 80°C) was added. Incubation was continued for 20 min. Reactions were terminated by the addition of SDS and EDTA. An aliquot of each reaction mixture was subjected to two-dimensional electrophoresis in 1.2% agarose. The gel was processed for in-gel hybridization, dried, and exposed to X-ray film. a, nicked pUC18; b, linear pUC18; c, positively supercoiled pUV18 topoisomers; d, relaxed pUC18; e, negatively supercoiled pUC18 topoisomers. 1st D, first dimension; 2nd D, second dimension.