Figure 1

Recombinant LigN is active as a DNA ligase. A. Schematic representation of Hfx.volcanii LigN protein structure showing the six structural domains that characterise this family of enzymes. B. Purified LigN, LigN-K139A and LigN-D141A proteins (lanes 1, 2 and 3 respectively) visualised by PAGE-Blue G90 staining following 10% SDS-PAGE. 5 μg of each protein was loaded per lane. The lane marked M contains molecular weight markers with molecular weights (in kDa) shown to the left. C. Adenylation assay. Purified LigN, LigN-K139A and LigN-D141A proteins (lanes 1, 2 and 3 respectively) were incubated with [³²P]NAD⁺, as described in the Methods, before being subjected to SDS-PAGE. Adenylated proteins were detected by autoradiography of the dried gel. D. Ligase activity assays carried out using λ Bst EII-digested DNA, as described in the Methods. Reactions were performed in 2.5 M KCl at 45°C with no added enzyme (lane 1, 10 minute incubation), LigN (lanes 2 – 5, incubation for 2.5, 5, 7.5 and 10 minutes), LigN-K139A (lane 6, 10 minute incubation) and LigN-K141A (lane 7, 10 minute incubation). E. Purified LigN and LigN-ΔC visualised by PAGE-Blue G90 staining following 10% SDS-PAGE. 5 μg of each protein was loaded per lane. The lane marked M contains molecular weight markers with molecular weights (in kDa) shown to the left. F. Adenylation assay. Purified LigN (lane 1) and LigN-ΔC (lane 2) were incubated with [³²P]NAD⁺, as described in the Methods, before being subjected to SDS-PAGE. Adenylated proteins were detected by autoradiography of the dried gel. G. DNA ligase activity assays performed as described in Methods without added enzyme (lane 1) or using LigN (lane 2) or LigN-ΔC (lane 3). LigN-ΔC was inactive in the assay.