High efficiency detection of bi-stranded abasic clusters in gamma-irradiated DNA by putrescine.
Bi-stranded abasic clusters, an abasic (AP) site on one DNA strand and another nearby AP site or strand break on the other, have been quantified using Nfo protein from Escherichia coli to produce a double-strand break at cluster sites. Since recent data suggest that Nfo protein cleaves inefficiently at some clusters, we tested whether polyamines, which also cut at AP sites, would cleave abasic clusters at higher efficiency. The data show that Nfo protein cleaves poorly at clusters containing immediately opposed AP sites and those separated by 1 or 3 bp. Putrescine (PUTR) cleaved more efficiently than spermidine or spermine, and did not cleave undamaged DNA. It cleaved abasic clusters in oligonucleotide duplexes more effectively than Nfo protein, including immediately opposed or closely spaced clusters. PUTR cleaved more efficiently than Nfo protein by a factor of approximately 1.7 or approximately 2 for DNA that had been gamma-irradiated in moderate or non-radioquenching conditions, respectively. This suggests that the DNA environment during irradiation affects the spectrum of cluster configurations. Further comparison of PUTR and Nfo protein cleavage may provide useful information on abasic cluster levels and configurations induced by ionizing radiation.
Georgakilas
Alexandros G
AG
Biology Department, Building 463, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.
Bennett
Paula V
PV
Sutherland
Betsy M
BM
D001080:Apurinic Acid / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005720:Gamma Rays; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011700:Putrescine / Q000378:metabolism*
D004279:DNA, Viral; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D001080:Apurinic Acid; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011700:Putrescine