Figure 2.

Template-independent synthesis by pol mu on immobilized DNA substrates distributed at low density on agarose beads. (A) Streptavidin agarose beads were used to immobilize two 73 bp DNA substrates with 3′ overhangs. ‘B' designates the 3′-biotin group of the substrate. An asterisk indicates the position of the radioisotope label. (B) In each reaction, 20 nM substrate was incubated with the protein(s) indicated above each lane in a 20 μl reaction for 1 h at 37°C. After incubation, reactions were heated at 100°C for 5 min to disrupt the biotin–streptavidin interaction, and then deproteinized and analyzed using 11% denaturing PAGE. Protein concentrations: Ku, 50 nM; X4-LIV, 100 nM; pol mu, 1.25 μM; dNTP, 5 mM. No ATP was added.