Mutagenesis, Vol. 15, No. 2, 115-120,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Characteristics of UV-induced repair patches relative to the nuclear skeleton in human fibroblasts
MGC Department of Radiation Genetics and Chemical Mutagenesis, LUMC, Leiden University, Leiden, The Netherlands
We have tried to characterize the nucleotide excision repair (NER) events associated with the nuclear skeleton in both repair-proficient and repair-deficient human cell lines following UV irradiation. The repair patches were labelled with biotin-16-dUTP and the repair sites were visualized by fluorescence microscopy using fluorescence-conjugated antibodies to biotin. The intensities of repair labelling measured for the three human cell lines of normal, 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 repair (TCR)-defective Cockayne syndrome cells (6-fold reduction after 1 h post-UV incubation). The intensity of repair labelling remained the same in TCR-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 syndrome cells, but not in XP-C cells. The tight association of repair patches generated by the TCR pathway with the nucleoskeleton in XP-C cells reinforces the concept of functional compartmentalization of the nucleus, where NER is highly heterogeneous.
1 To whom correspondence should be addressed at: Department of Radiation Genetics and Chemical Mutagenesis, Sylvious Laboratory, State University of Leiden, PO Box 9503, 2300 RA Leiden, The Netherlands. Tel: +31 7152 76164; Fax: +31 7152 21615; Email: natarajan{at}rullf2.medfac.leidenuniv.nl
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