Figure 1.

Purity and DNA synthetic activity of human Polι, Polη, Polκ, and Polβ. (A) Purified native and N-terminal GST-fusion Polι, Polη, Polκ, and native Polβ were analyzed on an 8% denaturing polyacrylamide gel and stained with Coomassie blue. (Lane 1) Molecular weight standards containing 500 ng of protein in each band; (lane 2) 300 ng of Polι; (lane 3) 500 ng of GST-Polι; (lane 4) 300 ng of Polη; (lane 5) 500 ng of GST-Polη; (lane 6) 500 ng of Polκ; (lane 7) 500 ng of GST-Polκ; (lane 8) 300 ng of Polβ. (B) The DNA synthetic activity of purified GST-fusion and native Polι, Polη, Polκ, and native Polβ was assayed using a 75-nt template oligonucleotide primed with a 40-nt 5′-³²P-labeled oligonucleotide (S1 substrate) in the presence of all four deoxynucleotides (100 μM) in buffer A containing 40 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 1 mM dithiothreitol, 100 μg/mL bovine serum albumin, and 10% glycerol. The DNA substrate (20 nM) was incubated with 2 nM of DNA polymerase at 37°C for 10 min. The reaction products were analyzed on an 8 M urea, 8% polyacrylamide gel, and the DNA bands were visualized by autoradiography.