Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases.
A synthetic procedure has been developed by which stable abasic sites are introduced into oligodeoxynucleotides at any desired position in the sequence. A modified tetrahydrofuran moiety, isosteric with 2'-deoxyribofuranose, serves as a structural analog of the natural apurinic/apyrimidinic site. We have also prepared oligodeoxynucleotides that lack cyclic structure at the abasic site but retain the carbon atoms of the phosphodiester backbone. These synthetic oligodeoxynucleotides are cleaved on the 5' side of the abasic site by endonuclease IV and by exonuclease III; they serve also as templates for avian myeloblastosis virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow fragment), and calf thymus DNA polymerase-alpha. Extension of primed templates by these DNA polymerases is blocked initially at the position immediately 3' to the abasic site; nucleoside monophosphates are subsequently incorporated opposite the lesion. The nucleotide most frequently incorporated opposite all abasic sites, regardless of structure, is dAMP. Significant "readthrough" at the abasic site was observed in experiments using avian myeloblastosis virus reverse transcriptase and DNA polymerase-alpha and, to a much lesser degree, with DNA polymerase I. We conclude that a modified tetrahydrofuran group can serve as a stable structural analog of 2'-deoxyribose in the apurinic/apyrimidinic site. These modified oligodeoxynucleotides should prove useful for studies of chemical mutagenesis.
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase