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 thiol groups of proteins at (or near) nascent repair sites in normal and xeroderma pigmentosum (group C) human cells. The reactive groups include cysteine 110 of histone H3, which is exposed in unfolded nucleosomes. Immediately after UV irradiation and a short pulse labeling of repair patches, intact nuclei were digested with restriction enzymes to release approximately 18% of the chromatin into soluble fragments, which are enriched (approximately 4-fold) in a constitutively transcribed gene. Upon organomercurial affinity fractionation, approximately 1.8% of the soluble chromatin remains bound in high salt (0.5 M NaCl) and is released with dithiothreitol. In normal cell chromatin, this fraction is enriched in nascent repair patches (1.5-1.8-fold) over the unbound fraction. This enrichment decreases following short chase periods with a time course similar to the loss of enhanced nuclease sensitivity of these regions (t 1/2 approximately 30 min). Much less enrichment of nascent repair patches is observed in the thiol-reactive fraction from XPC cells, which repair primarily the transcribed strand of active genes. These results suggest that transient nucleosome unfolding occurs during nucleotide excision repair in normal human cells, and this unfolding may require the XPC protein.
Baxter
B K
BK
Department of Biochemistry and Biophysics, P.O. Box 644660, Washington State University, Pullman, Washington 99164-4660, USA.
Smerdon
M J
MJ
D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D053938:DNA Fragmentation; D004260:DNA Repair; D006801:Humans; D009707:Nucleosomes / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014158:Transcription, Genetic; D014983:Xeroderma Pigmentosum / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
D009707:Nucleosomes; D013438:Sulfhydryl Compounds