Isolation of 8-methoxypsoralen accessible DNA domains from chromatin of intact cells.
The chromatin organization of living mammalian cells was probed using 8-methoxypsoralen (MOP). In intact cells, MOP intercalates into DNA domains which are also preferentially accessible to micrococcal nuclease. After UV365 nm irradiation of MOP-treated cells, this chemical forms bifunctional adducts crosslinking the two strands of DNA. Following extraction of cellular DNA, heat denaturation and renaturation at low temperature, the fraction of crosslinked DNA is obtained following enzymatic hydrolysis of unhybridized, non-crosslinked DNA by nuclease S1 treatment. An application of this procedure in the isolation of 8-methoxypsoralen-accessible DNA domains during DNA excision repair is shown.
Mathis
G A
GA
University of Zürich-Tierspital, Institute of Pharmacology and Biochemistry, Switzerland.
Althaus
F R
FR
D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000032:analysis* / Q000648:ultrastructure; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008099:Liver / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D008730:Methoxsalen / Q000302:isolation & purification* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D051381:Rats
D002843:Chromatin; D004247:DNA; D015253:Deoxyribonucleases, Type I Site-Specific; D008730:Methoxsalen