Figure 4.

Recombinant LigD2 and LigD1 POL domains. (A) Aliquots (7 µg) of the Ni-agarose preparations of the POL domains of AtuLigD2 and AtuLigD1 were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown in the left panel. Polymerase reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 5 mM MnCl₂, 100 µM rNTPs or dNTPs as specified, 50 nM 5′ ³²P-labeled 12-mer/24-mer DNA primer-template (depicted at bottom) and 1 µg of LigD2 or LigD1 POL as specified were incubated at 37°C for 20 min. The products were analyzed by denaturing PAGE. An autoradiograph of the gel is shown in the right panel. POL was omitted from the control reaction in lane −. (B) Glycerol gradient sedimentation was performed as described under Experimental Procedures. Aliquots (15 µl) of the odd-numbered fractions were analyzed by SDS-PAGE (top panel). The catalase, BSA, LigD2 POL and cytochrome c polypeptides are indicated. Polymerase activity was gauged by incubating 20 µl reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 5 mM MnCl₂, 100 µM rNTPs, 1 pmol 5′ ³²P-labeled 12-mer/24-mer DNA primer-template and 2 µl of the indicated gradient fractions at 37°C for 20 min. PAGE analysis of the primer extension products is shown in the bottom panel. (C) Metal specificity. Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 100 µM rNTPs, 50 nM 5′ ³²P-labeled 12-mer/24-mer DNA primer-template, 156 nM LigD2 POL, and either no divalent cation (–) or 5 mM Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺ or Zn²⁺ were incubated at 37°C for 20 min.