A requirement of FancL and FancD2 monoubiquitination in DNA repair.
The rare hereditary disorder Fanconi anemia (FA) can be caused by mutations in components of the FA core complex (FancA/B/C/E/F/G/L/M), a key regulator FancD2, the breast cancer susceptibility protein BRCA2/FancD1, or the newly identified FancJ/BRIP1 helicase. By performing yeast two-hybrid (Y2H) screens using N-terminal chicken (ch) FancD2 as a bait, we have identified chFancL, the likely ubiquitin E3 ligase subunit of the FA core complex. We also found that ectopically expressed FancD2 and FancL co-immunoprecipitated in 293T cells, and this interaction was dependent on the PHD domain of FancL. FANCL-disrupted chicken DT40 cells displayed defects in both FancD2 monoubiquitination and focus formation. Importantly, cell lines lacking the FANCL or FANCD2 genes, or carrying a "knock-in" mutation of the FancD2 monoubiquitination site (where the Lys 563 residue is changed to Arg), displayed quantitatively identical defects in the repair of I-SceI-induced chromosomal breaks by homologous recombination (HR). These data establish the role of FANCL and FancD2 monoubiquitination in HR repair.
Seki
Sohsuke
S
Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan.
Ohzeki
Mioko
M
Uchida
Akiko
A
Hirano
Seiki
S
Matsushita
Nobuko
N
Kitao
Hiroyuki
H
Oda
Tsukasa
T
Yamashita
Takayuki
T
Kashihara
Naoki
N
Tsubahara
Akio
A
Takata
Minoru
M
Ishiai
Masamichi
M
D000818:Animals; D002460:Cell Line; D002645:Chickens; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D005199:Fanconi Anemia / Q000235:genetics / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052240:Fanconi Anemia Complementation Group L Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism
D052236:Fanconi Anemia Complementation Group D2 Protein; D011994:Recombinant Proteins; D025801:Ubiquitin; D052240:Fanconi Anemia Complementation Group L Protein