Figure 3

Incisions at single nucleotide extrahelical loops by SP I^wt and SP I^H135A. (A) Autoradiogram of a denaturing PAGE. Lanes 1–2, intact substrates with no endonuclease treatment; lane 3, control oligonucleotide corresponding to the product of an incision 3' of the mismatched base; lanes 4–5, A and G extrahelical loop substrates incubated with CEL nuclease purified from celery; lanes 6–9, A and G extrahelical loop substrates incubated with recombinant SP I^wt and SP I^H135A nucleases; lanes 10–11, perfect duplex substrate incubated with SP I^wt and SP I^H135A nucleases, respectively. I, full-length oligonucleotide substrate labeled at the top strand. II, products of an incision at the mismatched nucleotide. The lower molecular weight bands at the bottom of the gel are mononucleotides and short oligonucleotides resulting from the 5' to 3' exonuclease activity of the native and recombinant enzymes [7]. (B) Design of a perfect duplex substrate, mismatched heteroduplex substrates and a control oligonucleotide corresponding to the CEL I reaction product. The location of the ³²P label is shown with an asterisk.