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 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.
Karmakar
P
P
MGC Department of Radiation Genetics and Chemical Mutagenesis, LUMC, Leiden University, Leiden, The Netherlands.
Natarajan
A T
AT
D002467:Cell Nucleus / Q000528:radiation effects*; D002478:Cells, Cultured; D003057:Cockayne Syndrome; D004260:DNA Repair; D005347:Fibroblasts / Q000528:radiation effects*; D006801:Humans; D008856:Microscopy, Fluorescence; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000528:radiation effects; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum
D018809:Proliferating Cell Nuclear Antigen