Characteristics of UV-induced repair patches relative to the nuclear skeleton in human fibroblasts

We have tried to characterize the nucleotide excision repair (NER) events a ssociated with the nuclear skeleton in both repair-proficient and repair-de ficient human cell lines following UV irradiation. The repair patches were labelled with biotin-16-dUTP and the repair sites were visualized by fluore scence microscopy using fluorescence-conjugated antibodies to biotin. The i ntensities of repair labelling measured for the three human cell lines of n ormal, xeroderma pigmentosum group C (XP-C) and Cockayne syndrome group B ( CS-B) are in good agreement with their known repair capabilities, Digestion of nuclei with DNase I markedly solubilized the repair patches in normal ( 3-fold reduction after 1 h post-UV incubation) and transcription-coupled re pair (TCR)-defective Cockayne syndrome cells (6-fold reduction after 1h pos t-UV incubation). The intensity of repair labelling remained the same in TC R-proficient XP-C cells after DNase I digestion, indicating that the repair events mediated by the TCR pathway are tightly associated with the nuclear skeleton, Treatment with ammonium sulphate after DNase I digestion further reduced the intensity of repair patches in both normal and Cockayne syndro me cells, but not in XP-C cells, The tight association of repair patches ge nerated by the TCR pathway with the nucleoskeleton in XP-C cells reinforces the concept of functional compartmentalization of the nucleus, where NER i s highly heterogeneous.