Figure 2.

Purity and active site titrations of LIG3 isoforms. A, 12% SDS-PAGE gel containing 0.5 μg of purified LIG3α and LIG3β with molecular masses of 102.7 and 95.9 kDa. M, protein sizing standards; α, Lig3α; β, Lig3β. B and C, representative active site titrations of LIG3α and LIG3β isoform, inset gel shows product (upper bands) and substrate (lower bands). In the absence of ATP, the adenylylated enzyme is limited to a single turnover ligation. Reactions contained 150 nm 28-mer nicked DNA substrate and 20 mm Mg²⁺ (see “Experimental procedures” for details). The equivalence point of the titration (denoted by arrow) indicates the concentration of adenylylated enzyme (data points are the means ± S.D.; n = 2).