BRCA1 ubiquitinates RPB8 in response to DNA damage.
The breast and ovarian tumor suppressor BRCA1 catalyzes untraditional polyubiquitin chains that could be a signal for processes other than proteolysis. However, despite intense investigations, the mechanisms regulated by the enzyme activity remain only partially understood. Here, we report that BRCA1-BARD1 mediates polyubiquitination of RPB8, a common subunit of RNA polymerases, in response to DNA damage. A proteomics screen identified RPB8 as a protein modified after epirubicin treatment in BRCA1-dependent manner. RPB8 interacted with BRCA1-BARD1 and was polyubiquitinated by BRCA1-BARD1 in vivo and in vitro. BRCA1-BARD1 did not destabilize RPB8 in vivo but rather caused an increase in the amount of soluble RPB8. Importantly, RPB8 was polyubiquitinated immediately after UV irradiation in a manner sensitive to BRCA1 knockdown by RNA interference. Substitution of five lysine residues of RPB8 with arginine residues abolished its ability to be ubiquitinated while preserving its polymerase activity. HeLa cell lines stably expressing this ubiquitin-resistant form of RPB8 exhibited UV hypersensitivity accompanied by up-regulated caspase activity. Our findings suggest that ubiquitination of a common subunit of RNA polymerases is a mechanism underlying BRCA1-dependent cell survival after DNA damage.
Wu
Wenwen
W
Division of Breast and Endocrine Surgery, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan.
Nishikawa
Hiroyuki
H
Hayami
Ryosuke
R
Sato
Ko
K
Honda
Akeri
A
Aratani
Satoko
S
Nakajima
Toshihiro
T
Fukuda
Mamoru
M
Ohta
Tomohiko
T
D019313:BRCA1 Protein / Q000378:metabolism*; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015251:Epirubicin / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D021122:Protein Subunits; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014452:Ubiquitins / Q000096:biosynthesis; D014466:Ultraviolet Rays
D019313:BRCA1 Protein; D021122:Protein Subunits; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D015251:Epirubicin; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases; D012321:DNA-Directed RNA Polymerases