A spin-labeled abasic DNA substrate for AP endonuclease

We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it intera cts with AP endonuclease, the key enzyme in DNA abasic site repair. The spi n labeled 23-mer with a U at position 12 of the upper strand is processed b y uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, A-P endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm. that the disposition of the bases immediately upstream of the abasic site is pa rticularly critical for cleavage by A-P endo, and establish that DNA-protei n interactions in this important enzyme can be examined using spin-labeled substrates. (C) 2001 Academic Press.