Figure 4.

hpol η-mediated postlesion full-length and single-nucleotide insertion assays using 1,N²-ε-G in template 5′-T(εG)A-3′ (1) and Primer_2. PAGE (20%, 7 M urea): A, full-length extension assay: hpol η (120 nM) elongated Primer_2 opposite G, and 1,N²-ε-G-containing DNA templates in the presence of a mixture of dNTPs (500 μM). All reactions were done at 37 °C for 5-, 30-, 60-, and 120-min (time gradients indicated with wedges). Lanes: 1 to 5 for unmodified template; 6 to 10 for 1,N²-ε-G-modified template. Single-nucleotide insertion assays: hpol η (10 nM) was incubated with B, Primer_2/control template 1, and C, Primer_2/1,N²-ε-G modified template 1 (5′-T(εG)A-3′), as well as individual dNTPs (100 μM). Lanes: 1 to 3 for dATP, 4 to 6 for dCTP, 7 to 9 for dGTP, 10 to 12 for dTTP. All reactions were done at 37 °C for 5-, 10-, and 30-min. P indicates the FAM-labeled Primer_2. See Experimental procedures and Table S1 for the oligonucleotide sequences used.