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 interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, AP 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 particularly critical for cleavage by AP endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.
Kolaczkowski
S V
SV
Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, USA.
Perry
A
A
Mckenzie
A
A
Johnson
F
F
Budil
D E
DE
Strauss
P R
PR
D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004578:Electron Spin Resonance Spectroscopy; D018583:Pliability; D013113:Spin Labels; D013379:Substrate Specificity
D013113:Spin Labels; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase