title: DHPLC and MS studies of a photoinduced intrastrand cross-link in DNA labeled with 5-bromo-2'-deoxyuridine.
abstract: It is well known that the replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) in DNA sensitizes it to UVB light. Irradiation of a biopolymer substituted in such a way leads to manifold kinds of DNA damage, such as intrastrand cross-links, single- and double-strand breaks or alkali-labile sites that were studied in the past with a broad spectrum of analytical methods. Here, we demonstrate that completely denaturing high-performance liquid chromatography (DHPLC), underestimated so far in DNA damage studies, could act as an inexpensive, and high-resolution substitute for the commonly employed gel electrophoresis. We report on the DHPLC/mass spectrometry (MS) analyses of photolytes obtained with the UV irradiation of aqueous solutions containing 40 base pairs of a long, double-stranded oligonucleotide labeled with BrdU in one of its strands. The UV-product was detected by HPLC at a temperature of 70°C. Subsequent MS analysis with electrospray ionization (ESI-MS) of the photolyte, enzymatic digestion of the irradiated material and HPLC and MS analysis (LC-MS) of the digest demonstrated unequivocally that an intrastrand covalent dimer, involving adenine and uracil, is formed in the irradiated system.
authors.affiliation: Department of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Wiczk
authors.affiliation: Department of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Miloch
authors.affiliation: Department of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. Electronic address: janusz@raptor.chem.univ.gda.pl.
authors.forename: Janusz
authors.initials: J
authors.lastname: Rak
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D001976:Bromouracil / Q000737:chemistry* / Q000528:radiation effects*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000528:radiation effects*; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects*; D003850:Deoxyribonuclease I / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry / Q000528:radiation effects; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry; D010782:Photolysis; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D001976:Bromouracil; D004247:DNA; D003850:Deoxyribonuclease I; D000469:Alkaline Phosphatase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010727:Phosphoric Diester Hydrolases
keywords: DNA UV damage; Damage quantum yield; Gel electrophoresis; Tandem mass spectrometry