Figure 1.

Strategy for the construction of single-stranded DNA. Gapped-duplex intermediates were produced as previously described (20, 24). The gapped strand of the duplex was provided by a plasmid that was grown in a wild-type strain, whereas the complementary strand contained several uracil residues as the corresponding plasmid was propagated in a dut, ung strain (CJ 236) (25). Single AAF-adducted oligonucleotides were ligated into the gap, and the covalently closed circular plasmid was purified from CsCl equilibrium sedimentation. The uracil-containing strand was then selectively degraded using an enzymatic mixture containing uracil DNA glycosylase, and the 3′ → 5′ exonuclease of exonucleaseIII and T7 DNA polymerase (25). Constructions containing the G-AAF adduct (G*) within the sequence 5′-GGG*- or 5′-GGCG*CC- were made along with the corresponding control vectors (no adduct).