Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo.
In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro.
Rosa
S
S
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanita, Rome, Italy.
Fortini
P
P
Karran
P
P
Bignami
M
M
Dogliotti
E
E
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D006898:Hydroxylamines; D009841:Oligonucleotides; D004247:DNA; C005214:methoxyamine; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase