Chromosoma

, Volume 119, Issue 6, pp 637–649

FANCJ/BRIP1 recruitment and regulation of FANCD2 in DNA damage responses

  • Fan Zhang
  • Qiang Fan
  • Keqin Ren
  • Arleen D. Auerbach
  • Paul R. Andreassen
Research Article

DOI: 10.1007/s00412-010-0285-6

Cite this article as:
Zhang, F., Fan, Q., Ren, K. et al. Chromosoma (2010) 119: 637. doi:10.1007/s00412-010-0285-6

Abstract

FANCJ/BRIP1 encodes a helicase that has been implicated in the maintenance of genomic stability. Here, to better understand FANCJ function in DNA damage responses, we have examined the regulation of its cellular localization. FANCJ nuclear foci assemble spontaneously during S phase and are induced by various stresses. FANCJ foci colocalize with the replication fork following treatment with hydroxyurea, but not spontaneously. Using FANCJ mutants, we find that FANCJ helicase activity and the capacity to bind BRCA1 are both involved in FANCJ recruitment. Given similarities to the recruitment of another Fanconi anemia protein, FANCD2, we tested for colocalization of FANCJ and FANCD2. Importantly, these proteins show substantial colocalization, and FANCJ promotes the assembly of FANCD2 nuclear foci. This process is linked to the proper localization of FANCJ itself since both FANCJ and FANCD2 nuclear foci are compromised by FANCJ mutants that abrogate its helicase activity or interaction with BRCA1. Our results suggest that FANCJ is recruited in response to replication stress and that FANCJ/BRIP1 may serve to link FANCD2 to BRCA1.

Supplementary material

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Fig. S1

DNA damage-induced assembly of FANCJ foci is influenced by ATR. a Immunoblots for ATR and FANCJ in extracts from untreated MCF7 cells transfected with siATR or a control (siGFP). The levels of actin protein were detected as a loading control. b The percentage of MCF7 cells transfected with a control siRNA or with siATR that displayed five or more FANCJ foci. Cells were left untreated or were exposed to 0.5 μM MMC for 20 h or were fixed at 7 h after exposure to 5 Gy IR. Values represent the average of three counts of 150 or more cells each ± standard deviation. Differences in the level of FANCJ foci in MMC-treated cells, with or without depletion of ATR, were statistically significant (p < 0.01) (GIF 24 kb)

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Fig. S2

Retroviral expression of FANCJ corrects G2-M accumulation in FA-J cells exposed to melphalan. Accumulation in G2-M following treatment with DNA interstrand crosslinkers is characteristic of FA cells. Histograms of DNA content were obtained by flow cytometry and comparable cell cycle profiles were observed for untreated EUFA30 fibroblasts expressing FANCJ or containing the empty pOZ-C vector. Cells were treated with melphalan for 48 h or were left untreated. The percentage of cells distributed to G2-M is indicated in each histogram. (GIF 20 kb)

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Fig. S3

Examples of the assembly of FANCJ foci in FA-J cells reconstituted with each form of FANCJ. Representative images of epitope-tagged FANCJ, detected with an antibody against the Flag peptide, for EUFA30 FA-J cells that contained the pOZ vector alone or which were reconstituted with FANCJ-S990A, FANCJ-K52R, or FANCJ-WT. Cells were exposed to 0.5 μM MMC for 20 h. An enlargement of the nucleus, which is positive for FANCJ foci in cells reconstituted with FANCJ-WT, is in the inset. The percentage of cells containing vector, FANCJ-S990A, or FANCJ-K52R that assembled foci was smaller than for cells reconstituted with FANCJ-WT and therefore representative fields do not show any positive cells for these lines. (GIF 35 kb)

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Fig. S4

Quantification of FANCJ and BRCA1 foci in FA-D2 cells and their corrected counterparts. Cells were exposed to 0.5 μM MMC for 20 h. Each value represents the average of three counts of 150 or more cells ± standard deviation. (GIF 13 kb)

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Fig. S5

FANCJ is required for the assembly of FANCD2 foci after exposure to IR or HU. FANCJ was depleted in MCF-7 cells using a siRNA, as shown in Fig. 6. As compared with cells transfected with a control siRNA (siGFP), depletion of FANCJ inhibited the assembly of FANCD2 foci following treatment with 2 mM HU or 10 Gy IR 7 h after exposure. The percentage of cells with five or more nuclear FANCD2 foci was calculated from three counts of 150 or more cells each and displayed ± standard deviation. Differences in the percentage of control cells or those depleted of FANCJ which displayed FANCD2 foci were statistically significant in cells exposed to IR or HU (p < 0.01). (GIF 10 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Fan Zhang
    • 1
  • Qiang Fan
    • 1
  • Keqin Ren
    • 1
  • Arleen D. Auerbach
    • 2
  • Paul R. Andreassen
    • 1
    • 3
  1. 1.Division of Experimental Hematology and Cancer BiologyCincinnati Children’s Research FoundationCincinnatiUSA
  2. 2.Laboratory of Human Genetics and HematologyThe Rockefeller UniversityNew YorkUSA
  3. 3.Department of PediatricsUniversity of Cincinnati College of MedicineCincinnatiUSA

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