Kinetics and mechanism of DNA repair. Evaluation of caged compounds for use in studies of u.v.-induced DNA repair

• Published in: Biochemical journal Vol. 266; no. 3; pp. 891 - 895
• Main Authors: Meldrum, R A, Shall, S, Wharton, C W
• Format: Journal Article
• Language: English
• Published: England 15.03.1990
• Subjects: Animals; Cell Line; Cricetinae; Cricetulus; Deoxyribonucleosides; DNA - isolation & purification; DNA - radiation effects; DNA Damage; DNA Repair; Humans; Kinetics; Lasers; Photolysis; Spectrophotometry, Ultraviolet; Ultraviolet Rays
• ISSN: 0264-6021; 1470-8728

Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the gamma-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released from the cage by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside triphosphate is isotopically labelled in the alpha-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair.