Figure 8. Cross-links containing the abasic site in the template strand block primer extension by ϕ29 DNA polymerase extension, while an un-cross-linked abasic site in the template strand is a partial block.

The ³²P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) 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 60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′-³²P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA); lanes 2 and 3 are Maxam-Gilbert G- and A+G-reactions carried out on the 5′-³²P-labeled full-length extension product; lane 4 is the 15 nt, 5′-³²P-labeled primer; lane 5 is the 5′-³²P-labeled full-length extension product; lane 6, primer extension on the single-strand substrate H containing dU in the template; lane 7, single-strand substrate I containing Ap in the template; lane 8, duplex substrate J containing dU in the template; lane 9, duplex substrate K containing an un-cross-linked Ap site in the template strand; lane 10, duplex substrate L containing reduced dG-Ap cross-link 5 with the Ap residue in the template strand; lane 11, duplex substrate X containing the dA-Ap cross-link in which the Ap residue is in the template strand.