Characterization of Biotinylated Repair Regions in Reversibly Permeabilized Human Fibroblasts

Abstract

We have examined the incorporation of biotinyl-11-deoxyuridine triphosphate (BiodUTP) into excision repair patches of UV-irradiated confluent human fibroblasts. Cells were reversibly permeabilized to BiodUTP with lysolecithin, and biotin was detected in DNA on nylon filters using a streptavidin/alkaline phosphatase colorimetric assay. Following a UY dose of 12 J/m2, maximum incorporation of BiodUTP occurred at a lysolecithin concentration (80-100 μg/mL) similar to that for incorporation of dTTP. Incorporation of BiodUTP into repair patches increased with UV dose up to 4 and 8 J/m2in two normal human fibroblast strains, while no incorporation of BiodUTP was observed in xeroderma pigmentosum (group A) human fibroblasts. The repair-incorporated biotin was not removed from the DNA over a 48-h period, and only slowly disappeared after longer times (~30% in 72 h), while little of the biotiji remained in cells induced to divide. Furthermore, the stability of the biotin in repaired DNA was unaffected by a second dose of UV radiation several hours after the biotin-labeling period to induce a “second round” of excision repair. Exonuclease III digestion and gap-filling with DNA polymerase I indicate that the majority of biotin-labeled repair patches (~80%) are rapidly ligated in confluent human cells. However, the remaining patches were not ligated after a 24-h chase period, in contrast to dTTP-labeled repair patches. The BiodUMP repair label in both chromatin and DNA is preferentially digested by staphylococcal nuclease, preventing the use of this enzyme for nucleosome mapping in these regions. However, restriction enzyme and DNase I digestions of the isolated nuclei demonstrate that at least some of the repair-incorporated BiodUMP becomes associated with nucleosome core DNA following nucleosome rearrangement. Therefore, the biotin tag does not appear to prevent the folding of nascent repair patches into native nucleosome structures. © 1992, American Chemical Society. All rights reserved.