A method for the isolation of cross-linked nucleosides from DNA: application to cross-links induced by nitrous acid.
A procedure is reported for the isolation of cross-linked nucleosides from nitrous acid-treated calf thymus DNA. Cross-linked DNA was hydrolyzed enzymatically with deoxyribonuclease I and snake venom phosphodiesterase and fractionated on a DEAE-Sephadex column. After desalting, the fractions were characterized by ultraviolet spectroscopy, anion exchange high pressure liquid chromatography, gel filtration, and two dimensional thin layer chromatography. A cross-linked dinucleotide, and a series of oligonucleotides were isolated. The oligomers, which had resisted digestion by the above enzyme system, were digested to the nucleoside level by a spleen phosphodiesterase-alkaline phosphatase combination. A second cross-linked product was isolated from this mixture. The cross-linked nucleosides were less than 0.17% of the total nucleotides of the DNA. The methods developed here are recommended for the isolation of products from DNA treated with other cross-linking agents.
D000469:Alkaline Phosphatase; D000818:Animals; D002417:Cattle; D004247:DNA; D003851:Deoxyribonucleases; D008722:Methods; D009608:Nitrous Acid / Q000494:pharmacology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000302:isolation & purification*; D010727:Phosphoric Diester Hydrolases; D012910:Snake Venoms; D013056:Spectrophotometry, Ultraviolet; D013154:Spleen; D013950:Thymus Gland / Q000032:analysis*
D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D012910:Snake Venoms; D004247:DNA; D003851:Deoxyribonucleases; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D009608:Nitrous Acid