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The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair

Abstract

BRIP1 (also called BACH1) is a DEAH helicase that interacts with the BRCT domain of BRCA1 (refs. 16) and has an important role in BRCA1-dependent DNA repair and checkpoint functions1,2,6,7. We cloned the chicken ortholog of BRIP1 and established a homozygous knockout in the avian B-cell line DT40. The phenotype of these brip1 mutant cells in response to DNA damage differs from that of brca1 mutant cells and more closely resembles that of fancc mutant cells, with a profound sensitivity to the DNA-crosslinking agent cisplatin and acute cell-cycle arrest in late S-G2 phase. These defects are corrected by expression of human BRIP1 lacking the BRCT-interaction domain. Moreover, in human cells exposed to mitomycin C, short interfering RNA–mediated knock-down of BRIP1 leads to a substantial increase in chromosome aberrations, a characteristic phenotype of cells derived from individuals with Fanconi anemia. Because brip1 mutant cells are proficient for ubiquitination of FANCD2 protein, our data indicate that BRIP1 has a function in the Fanconi anemia pathway that is independent of BRCA1 and downstream of FANCD2 activation.

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Figure 1: Generation of a homozygous brip1 knockout in the DT40 cell line.
Figure 2: Sensitivity of brip1 and brca1 mutant cells to DNA-damaging agents.
Figure 3: brip1 mutant cells are proficient for HDR of an I-SceI–induced DNA double-strand break.
Figure 4: brip1 cells, but not brca1 cells, arrest with a DNA content near 4N after exposure to cisplatin.
Figure 5: brip1 cells have increased chromosome instability.

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Acknowledgements

We thank D. Livingston for human BRIP1 cDNA; J. Sale for DT40 cells; J. Di Noia for DT40 cells with an integrated DR-GFP; K.J. Patel for antibody to FANCD2, fancc mutant cells and FANCC disruption construct; A. Oostra for assistance with chromosome breakage assays in HeLa cells; H. Joenje and J. de Winter for advice and for communicating data before publication; and our colleagues at the Laboratory of Molecular Biology for advice and technical assistance. C.J.V. was funded in part by a grant from the Association for International Cancer Research.

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Correspondence to Kevin Hiom.

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Supplementary information

Supplementary Fig. 1

Clustal alignment of human (Hs) and chicken (Gg) BRIP1 protein sequences. (PDF 118 kb)

Supplementary Table 1

Oligonucleotide primers used for PCR as described in Methods. (PDF 26 kb)

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Bridge, W., Vandenberg, C., Franklin, R. et al. The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair. Nat Genet 37, 953–957 (2005). https://doi.org/10.1038/ng1627

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