A novel hydrogen peroxide-induced phosphorylation and ubiquitination pathway leading to RNA polymerase II proteolysis.
DNA damage-induced ubiquitination of the largest subunit of RNA polymerase II, Rpb1, has been implicated in transcription-coupled repair for years. The studies so far, however, have been limited to the use of bulky helix-distorting DNA damages caused by UV light and cisplatin, which are corrected by the nucleotide excision repair pathway. Non-bulky, non-helix-distorting damages are caused at high frequency by reactive oxygen species in cells and corrected by the base excision repair pathway. Contrary to a classic view, we recently found that the second type of DNA lesions also causes RNA polymerase II stalling in vitro. In this paper, we show that hydrogen peroxide (H(2)O(2)) causes significant ubiquitination and proteasomal degradation of Rpb1 by mechanisms that are distinct from those employed after UV irradiation. UV irradiation and H(2)O(2) treatment cause characteristic changes in protein kinases phosphorylating the carboxyl-terminal domain at Ser-2 and -5. The H(2)O(2)-induced ubiquitination is likely dependent on unusual Ser-5 phosphorylation by ERK1/2. Moreover, the H(2)O(2)-induced ubiquitination occurs on transcriptionally engaged polymerases without the help of Cockayne syndrome A and B proteins and von Hippel-Lindau tumor suppressor proteins, which are all required for the UV-induced ubiquitination. These results suggest that stalled polymerases are recognized and ubiquitinated differentially depending on the types of DNA lesions. Our findings may have general implications in the basic mechanism of transcription-coupled nucleotide excision repair and base excision repair.
Inukai
Naoto
N
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology and PRESTO, Japan Science and Technology Agency, Yokohama 226-8501, Japan.
Yamaguchi
Yuki
Y
Kuraoka
Isao
I
Yamada
Tomoko
T
Kamijo
Sachiko
S
Kato
Junko
J
Tanaka
Kiyoji
K
Handa
Hiroshi
H
D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D004249:DNA Damage; D004260:DNA Repair; D005260:Female; D005347:Fibroblasts; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007680:Kidney Neoplasms; D048728:MAP Kinase Kinase Kinase 1; D048730:MAP Kinase Kinase Kinase 2; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D012319:RNA Polymerase II / Q000032:analysis / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism*; D014466:Ultraviolet Rays
D021122:Protein Subunits; D025801:Ubiquitin; D012694:Serine; D006861:Hydrogen Peroxide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D048730:MAP Kinase Kinase Kinase 2; D020930:MAP Kinase Kinase Kinases; C482576:MAP3K1 protein, human; C482618:MAP3K2 protein, human; D012319:RNA Polymerase II