BRCA1-independent ubiquitination of FANCD2.
Monoubiquitination of the FANCD2 protein is a key step in the Fanconi anemia (FA) tumor suppressor pathway, coinciding with this molecule's accumulation at sites of genome damage. Strong circumstantial evidence points to a requirement for the BRCA1 gene product in this step. Here, we show that the purified BRCA1/BARD1 complex, together with E1 and UbcH5a, is sufficient to reconstitute the monoubiquitination of FANCD2 in vitro. Although siRNA-mediated knockdown of BRCA1 in human cells results in defective targeting of FANCD2 to sites of DNA damage, it does not lead to a defect in FANCD2 ubiquitination. Furthermore, ablation of the RING finger domains of either BRCA1 or BARD1 in the chicken B cell line DT40 also leaves FANCD2 modification intact. Consequently, while BRCA1 affects the accumulation of FANCD2 at sites of DNA damage, BRCA1/BARD1 E3 ligase activity is not essential for the monoubiquitination of FANCD2.
Vandenberg
Cassandra J
CJ
Protein & Nucleic Acid Chemistry Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
Gergely
Fanni
F
Ong
Chong Yi
CY
Pace
Paul
P
Mallery
Donna L
DL
Hiom
Kevin
K
Patel
Ketan J
KJ
D000818:Animals; D019313:BRCA1 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D005199:Fanconi Anemia / Q000201:enzymology* / Q000235:genetics; D052236:Fanconi Anemia Complementation Group D2 Protein; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D033862:Iron-Binding Proteins; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
D019313:BRCA1 Protein; D002352:Carrier Proteins; C499905:FANCD2 protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D033862:Iron-Binding Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases