Sequence-dependent repair of synthetic AP sites in 15-mer and 35-mer oligonucleotides: role of thermodynamic stability imposed by neighbor bases.

We previously reported that 15-mer oligonucleotides with a central 1, N(6)-epsilonA were cleaved by alkylpurine-DNA N-glycosylase as a function of T(m), modulated by neighbor bases [Hang, B., Sági, J., and Singer, B. (1998) J. Biol. Chem. 273, 33406-33413]. This type of investigation has now been extended to cleavage by Escherichia coli endonuclease IV of a centrally placed synthetic AP site using both 15-mer and 35-mer duplexes. In 15-mers, the triplet sequences adjunct to the central AP site greatly affected the thermodynamic stability. The repair rate paralleled the thermal stability since endonuclease IV requires a double-stranded substrate. When the AP site-containing duplexes were 35-mers, there was also a general correlation between the thermostability and cleavage efficiency. However, the difference in the cleavage rates between different sequences was much less than with the 15-mers. Since the 35-mers were more than 96% annealed, this difference presumably results from local stability and structure adjacent to the AP site. These results suggest that under enzyme limiting conditions or overproduction of AP sites, sequence-dependent differential repair could occur in vivo.