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)