PSORALEN DAMAGE IN HUMAN CELLS: DNA REPAIR AND EFFECTS ON REPLICATION

Aspects of the damage, repair, and replication of DNA were measured following photoaddition by furocoumarins (psoralens) in SV40-transformed normal human, repair deficient xeroderma pigmentosum (XP) group A, and crosslink sensitive Fanconi's anemia (FA) cells.; 8-methoxypsoralen (8MOP) binding was twofold greater than angelicin (ANG) binding after exposure to 360 nm UV light (NUV). Saturation was observed by both compounds for light doses greater than 30,000 J/m('2) and a concentration of 10 (mu)g/ml.; DNA interstrand crosslinking by 8MOP, ANG, 5-methoxypsoralen (5MOP), 3-carbethoxypsoralen (3CP), and 5-geranoxypsoralen (5GYP) was determined by a novel density-shift method. Crosslink formation kinetics were linear for the NUV doses and concentrations used. Crosslink frequency following exposure to 25 (mu)g/ml 8MOP and 45,000 J/m('2) NUV was one crosslink per 10('7) daltons of chromatin. Crosslinking by 5MOP, ANG, 3CP, and 5GYP was 50%, 15%, 5%, and 0%, respectively, of that by 8MOP with identical treatment. This is the first indication of crosslinking by ANG and 3CP in human cells.; Alkaline instability of mitomycin (MMC) and nitrogen mustard (HN(,2)) crosslinks was indicated following akaline isopycnic gradient analysis.; Crosslinking inhibits repair of monoadducts and pyrimidine dimers in normal cells, and crosslink repair does not involve extensive precursor incorporation. Normal and FA cells repaired monoadducts. Lack of repair synthesis in XP cells suggested similarity between pyrimidine dimer repair and repair of psoralen lesions. Repair measurements showed a plateau in precursor incorporation following exposure to 7,500 J/m('2) of NUV and 15 (mu)g/ml greater than 8MOP or ANG. The repair synthesis plateau resulting from furocourmarin treatment indicated saturation of repair and was about 20% of that from an independent saturating dose of 254 nm UV light.; Normal cells removed most crosslinks within 24 hours with a t(, 1/2) of about 9 hours. No crosslink repair occurred in FA or XP cells over a 24 hour period. The results suggest that endonuclease incision is required for crosslink repair.; Inhibition of DNA synthesis after 8MOP or ANG treatment appears to be associated with blocks to replicon elongation caused by monoadducts rather than inhibition of replicon initiation.