Bk. Baxter et Mj. Smerdon, NUCLEOSOME UNFOLDING DURING DNA-REPAIR IN NORMAL AND XERODERMA-PIGMENTOSUM (GROUP-C) HUMAN-CELLS, The Journal of biological chemistry, 273(28), 1998, pp. 17517-17524
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.