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 UV dose of 12 J/m2, maximum incorporation of BioUTP occurred at a lysolecithin concentration (80-100 micrograms/mL) similar to that for incorporation of dTTP. Incorporation of BiodUTP into repair patches increased with UV dose up to 4 and 8 J/m2 in 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 (approximately 30% in 72 h), while little of the biotin 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 (approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

“…Although this range is close to the ϳ2% of total fragments in the S fraction trapped by organomercurial chromatography, it must be stressed that this calculation assumes that all labeled repair patches are unfolded (some for 30 min) and is an overestimate of the fraction of unfolded nucleosomes. 3 DNA Fragment Size of the Fractions-Treatment of nuclei with restriction enzymes can result in a nucleosomal repeat pattern of DNA fragments on a gel (29,34). However, restriction enzymes provide a single, site-specific cut in chromatin DNA without the exonuclease "trimming" associated with staphylococcal nuclease (see Ref.…”