Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways.
Fanconi anaemia (FA) is a chromosomal instability disorder characterized by cellular sensitivity to DNA interstrand crosslinking agents and a high risk of cancer. Six of the eight proteins encoded by the known FA genes form a nuclear complex which is required for the monoubiquitination of the FANCD2 protein. FANCD2 complexes and colocalizes with BRCA1, but its presumptive role in DNA repair has not yet been clearly defined. We used yeast two-hybrid analysis to test for interaction between FANCD2 and 10 proteins involved in homologous recombination repair. FANCD2 did not interact with RAD51, the five RAD51 paralogs, RAD52, RAD54 or DMC1. However, it bound to a highly conserved C-terminal site in BRCA2 that also binds FANCG/XRCC9. FANCD2 and BRCA2 can be coimmunoprecipitated from cell extracts of both human and Chinese hamster wild-type cells, thus confirming that the interaction occurs in vivo. Formation of nuclear foci of FANCD2 was normal in the BRCA2 mutant CAPAN-1 cells, which indicates that the recruitment of FANCD2 to sites of DNA-repair is independent of wild-type BRCA2 function. FANCD2 colocalized with RAD51 in foci following treatment with mitomycin C or hydroxyurea, and colocalized very tightly with PCNA after treatment with hydroxyurea. These findings suggest that FANCD2 may have a role in the cellular response to stalled replication forks or in the repair of replication-associated double-strand breaks, irrespective of the type of primary DNA lesion.
Hussain
Shobbir
S
Division of Genetics and Development, Guy's, King's and St Thomas's School of Medicine, King's College London, UK.
Wilson
James B
JB
Medhurst
Annette L
AL
Hejna
James
J
Witt
Emily
E
Ananth
Sahana
S
Davies
Adelina
A
Masson
Jean-Yves
JY
Moses
Robb
R
West
Stephen C
SC
de Winter
Johan P
JP
Ashworth
Alan
A
Jones
Nigel J
NJ
Mathew
Christopher G
CG
D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein; D006801:Humans; D047468:Immunoprecipitation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D051135:Rad51 Recombinase; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D024682:BRCA2 Protein; D004268:DNA-Binding Proteins; C499905:FANCD2 protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C495153:RAD51 protein, human; D051135:Rad51 Recombinase