Figure 6.

The dA-3’ddR5p cross-link blocks DNA replication by ϕ29 polymerase. The 15 nt ³²P-labeled primers were incubated with the DNA substrates, the polymerase enzyme (10 U), and the four dNTPs (1 mM in each) in Tris–HCl (50 mM, pH 7.5), MgCl₂ (10 mM), (NH₄)₂SO₄ (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/ml) for 30 min at 24°C. After reaction work-up, the primer extension products were analyzed on a 20% denaturing polyacrylamide gel. Lanes 1, 2 and 3 are Maxam–Gilbert G, A+G, and iron–EDTA–H₂O₂ cleavage reactions carried out on a 5’-³²P-labeled standard of the full-length extension product (5’-³²P-GAT CAC AGT GAG TAG AAT AGA ATA CCA GAT ACA TGA ACT TAG ACA TAT ACA CAG AT); lane 4 is the 5’-³²P-labeled full-length extension product; lane 5 is the 15 nt, 5’-³²P-labeled primer; lanes 6–11, primer extension on substrates M–R. Sequences of these substrates are shown in Supplementary Figure S6.