Functional TFIIH is required for UV-induced translocation of CSA to the nuclear matrix.
Transcription-coupled repair (TCR) efficiently removes a variety of lesions from the transcribed strand of active genes. Mutations in Cockayne syndrome group A and B genes (CSA and CSB) result in defective TCR, but the molecular mechanism of TCR in mammalian cells is not clear. We have found that CSA protein is translocated to the nuclear matrix after UV irradiation and colocalized with the hyperphosphorylated form of RNA polymerase II and that the translocation is dependent on CSB. We developed a cell-free system for the UV-induced translocation of CSA. A cytoskeleton (CSK) buffer-soluble fraction containing CSA and a CSK buffer-insoluble fraction prepared from UV-irradiated CS-A cells were mixed. After incubation, the insoluble fraction was treated with DNase I. CSA protein was detected in the DNase I-insoluble fraction, indicating that it was translocated to the nuclear matrix. In this cell-free system, the translocation was dependent on UV irradiation, CSB function, and TCR-competent CSA. Moreover, the translocation was dependent on functional TFIIH, as well as chromatin structure and transcription elongation. These results suggest that alterations of chromatin at the RNA polymerase II stall site, which depend on CSB and TFIIH at least, are necessary for the UV-induced translocation of CSA to the nuclear matrix.
Saijo
Masafumi
M
Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan. saijom@fbs.osaka-u.ac.jp
Hirai
Tamami
T
Ogawa
Akiko
A
Kobayashi
Aki
A
Kamiuchi
Shinya
S
Tanaka
Kiyoji
K
D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D002843:Chromatin / Q000502:physiology / Q000648:ultrastructure; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D015530:Nuclear Matrix / Q000378:metabolism*; D021381:Protein Transport / Q000528:radiation effects; D012319:RNA Polymerase II / Q000378:metabolism*; D051758:Transcription Factor TFIIH / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D002843:Chromatin; C095472:ERCC8 protein, human; D014157:Transcription Factors; D051758:Transcription Factor TFIIH; D012319:RNA Polymerase II; D045643:DNA Repair Enzymes