Mechanism of DNA cleavage and substrate recognition by a bovine apurinic endonuclease.
The location of the phosphodiester bond cleaved by homogeneous Mg2+-dependent apurinic endodeoxyribonuclease (EC 3.1.25.2; APE) of bovine calf thymus has been determined by using a 21-mer oligonucleotide containing a single central apurinic site as a substrate. A single product of cleavage consistent with cleavage of the oligonucleotide 5' to the apurinic site, and leaving a 3' hydroxyl group, was identified. This enzyme is, therefore, a class II apurinic endonuclease. The substrate specificities of this enzyme have been determined by using a variety of natural and synthetic DNAs or oligonucleotides containing base-free sites. Calf thymus APE has an absolute requirement for a double-stranded DNA and requires an abasic site as a substrate. The presence of a base fragment such as a urea residue, an alkoxyamine group attached to the C'-1 position of the abasic site, or reduction of the C'-1 aldehyde abolishes the APE activity of this enzyme. Synthetic abasic sites containing either ethylene glycol, propanediol, or tetrahydrofuran interphosphate linkages are excellent substrates for bovine APE. These results indicate that APE has no absolute requirement for either ring-opened or ring-closed deoxyribose moieties in its recognition of DNA-cleavage substrates. The enzyme may interact with the pocket in duplex DNA that results from the base loss or with the altered conformations of the phosphodiester backbone that result from the abasic site.
Sanderson
B J
BJ
Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Chang
C N
CN
Grollman
A P
AP
Henner
W D
WD
D000588:Amines / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004278:DNA, Superhelical / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
D000588:Amines; D004278:DNA, Superhelical; D009838:Oligodeoxyribonucleotides; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium