(A) Templated additions. Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 8.0), 1 pmol of 32P-labeled primer-template DNA substrate (shown at bottom), either no added nucleotide (lanes –), 100 µM of a single rNTP as specified, or a mixture of 100 µM each of ATP, GTP, CTP and UTP (AGCU), 200 ng MsmPolD2, and either 1.25 mM MgCl2, MnCl2, or CoCl2 as specified were incubated for 10 min at 37°C. (B) Nontemplated additions. Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 8.0), 1,25 mM CoCl2, 1 pmol of 32P-labeled single-strand primer (shown at bottom), either no added nucleotide (lanes –), 100 µM of a single rNTP as specified, or a mixture of 100 µM each of ATP, GTP, CTP and UTP (AGCU), and 200 ng MsmPolD2 were incubated for 10 min at 37°C. The reaction products were resolved by PAGE and visualized by autoradiography.