Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.
DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.
Legault
J
J
Unité de Sante et Environnement, Pavillon CHUL, Centre de Recherche du CHUQ et Université Laval, Sainte-Foy, Québec, Canada.
Tremblay
A
A
Ramotar
D
D
Mirault
M E
ME
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002384:Catalysis; D002843:Chromatin / Q000187:drug effects / Q000528:radiation effects; D004247:DNA / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D029968:Escherichia coli Proteins; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D008190:Lyases / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D013308:Streptonigrin / Q000494:pharmacology; D013713:Teniposide / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000378:metabolism; D014812:Vitamin K / Q000494:pharmacology
D000970:Antineoplastic Agents; D002843:Chromatin; D029968:Escherichia coli Proteins; D006490:Hemostatics; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D014812:Vitamin K; D013308:Streptonigrin; D004247:DNA; D013713:Teniposide; D008741:Methyl Methanesulfonate; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase