Characterization and evaluation by ³²P-postlabelling of psoralen-type DNA adducts in HeLa cells

Abstract

Samples of DNA irradiated at 405 and/or 365 nm in the presence of 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelling assay using three hydrolysis enzymes other than those employed previously. These enzymes (deoxyribonucleaseI, venom phosphodiesterase and alkaline phosphatase) liberated 3'-adducted dinucleotide monophosphate instead of the 5'-modified dinucleotide monophosphate normally obtained. The first separation chromatography (D1) of samples irradiated in the presence of 8-MOP showed a single spot above the origin, and the next separation (D2) resolved this spot into two components (spots I and II). Double irradiation experiments in which samples of DNA were first irradiated at 405 nm before being irradiated at 365 nm showed that spot II could be transformed into spot I. The use of 6,4,4'-trimethylangelicin, which induced only photomonoadducts under UVA irradiation, gave only spot II. These two results indicated that spots I and II were respectively due to interstrand cross-links and monoadducts. Dose-effect experiments showed that spots I and II were dose dependent, and low-dose irradiations permitted us to measure one interstrand cross-link and two monoadducts per 10(8) base pairs.

Equilenin‐2′‐deoxynucleoside adducts: analysis with nano‐liquid chromatography coupled to nano‐electrospray tandem mass spectrometry

“…Therefore, a system was elaborated using a column switching technique using a small pre-column (5 mm ð 300 µm i.d.) filled with reversed-phase material C 18 . This approach not only allowed the injection of a few microliters of sample but also allowed the selective capture of the analytes of interest on the pre-column.…”

Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

“…To characterize chemical structural change of nucleotides in DNA or to quantify the stucturally changed nucleoties, long DNA chains are often required to be digested into single nucleosides facilitating followed MS analysis. Nevertheless, the digestion of genomic DNA into single nucleosides was required at least three enzymes, involving deoxyribonuclease (e.g., DNase P1 or DNase I), phosphodiesterase (e.g., snake venom phosphodiesterase, SVP), and alkaline phosphatase (ALPase). ,− ,− DNase I are applied to cleave DNA into small oligonucleotides and mononucleotides, phosphodiesterases attack the 3′- or 5′-terminal OH-groups releasing 5′- and 3′-mononucleotides respectively and ALPase are responsible for removing phosphate from the mononucleotides. However, this most commonly used approach has encountered some critical problems.…”

Technologies for Detection of DNA Damage and Mutations

“…5.2). Variations of this approach have been used to evaluate cis-platin adducts in calf thymus DNA (Forsti and Hemrninki, 1994) and psoralen-type DNA adducts in HeLa cells (Gillardeaux et at., 1994), and UVphoto products (Bykov et al, 1995 [-y-32PJ-ATP (Redivue", 3000 Ci/mmole):obtained from Amersham Corporation (catalog # AA0066).…”