Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin.
We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.
Lorenz
J D
JD
Biochemistry/Biophysics Program, Washington State University, Pullman 99164-4660.
Watkins
J F
JF
Smerdon
M J
MJ
D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D006801:Humans; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D014466:Ultraviolet Rays
D008244:Lysophosphatidylcholines