NUCLEOSOME UNFOLDING DURING DNA-REPAIR IN NORMAL AND XERODERMA-PIGMENTOSUM (GROUP-C) HUMAN-CELLS

The fate of nucleosomes during nucleotide excision repair is unclear. We have used organomercurial chromatography to capture accessible thio l groups of proteins at (or near) nascent repair sites in normal and x eroderma pigmentosum (group C) human cells. The reactive groups includ e cysteine 110 of histone H3, which is exposed in unfolded nucleosomes . Immediately after UV irradiation and a short pulse labeling of repai r patches, intact nuclei were digested with restriction enzymes to rel ease similar to 18% of the chromatin into soluble fragments, which are enriched (similar to 4-fold) in a constitutively transcribed gene. Up on organomercurial affinity fractionation, similar to 1.8% of the solu ble chromatin remains bound in high salt (0.5 M NaCl) and is released with dithiothreitol. In normal cell chromatin, this fraction is enrich ed in nascent repair patches (1.5-1.8 fold) over the unbound fraction. This enrichment decreases following short chase periods with a time c ourse similar to the loss of enhanced nuclease sensitivity of these re gions (t 1/2 approximate to 30 min). Much less enrichment of nascent r epair patches is observed in the thiol-reactive fraction from XPC cell s, which repair primarily the transcribed strand of active genes. Thes e results suggest that transient nucleosome unfolding occurs during nu cleotide excision repair in normal human cells, and this unfolding may require the XPC protein.