Title
Effects of microinjected photoreactivity enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts
Author
Medisch Biologisch Laboratorium TNO
Roza, L.
Vermeulen, W.
Bergen Henegouwen, J.B.A.
Eker, A.P.M.
Jaspers, N.G.J.
Lohman, P.H.M.
Hoeijmakers, J.H.J.
Publication year
1990
Abstract
UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this differnece is preferential removal of some type(s) of nondimer lesions, e.g., (6-4)photoproducts, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.
Subject
Deoxyribodipyrimidine photolyase
Thymine
DNA
Lyase
Pyrimidine dimer
Cell culture
DNA damage
DNA repair
Human
Human cell
Priority journal
Xeroderma pigmentosum
DNA replication
Fibroblast
Kinetics
Metabolism
Microinjection
Radiation exposure
Radiation response
Reference value
Ultraviolet radiation
Cells, Cultured
Deoxyribodipyrimidine Photo-Lyase
DNA
Dose-Response Relationship, Radiation
Fibroblasts
Human
Kinetics
Lyases
Microinjections
Pyrimidine Dimers
Reference Values
Support, Non-U.S. Gov't
Ultraviolet Rays
Xeroderma Pigmentosum
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http://resolver.tudelft.nl/uuid:1e6676e8-130f-4600-9faa-85a92eeb48be
TNO identifier
231091
ISSN
0008-5472
Source
Cancer Research, 50 (50), 1905-1910
Document type
article