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FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

Castella M, Jacquemont C, Thompson EL, Yeo JE, Cheung RS, Huang JW, Sobeck A, Hendrickson EA, Taniguchi T - PLoS Genet. (2015)

Bottom Line: Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle.ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment.Additionally, BRCA1 was required for efficient FA core complex foci formation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

No MeSH data available.


Related in: MedlinePlus

BRCA1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs, left untreated or treated with MMC (60 ng/ml) for 24h. The number of foci/cell (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05 (Compared to siControl for each foci, respectively). (B) BRCA1-deficient HCC1937 and complemented cell lines were treated with MMC (60 ng/ml) for 24h. The percentage of cells with > 5 foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to HCC1937+BRCA1 for each foci, respectively).
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pgen.1005563.g006: BRCA1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs, left untreated or treated with MMC (60 ng/ml) for 24h. The number of foci/cell (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05 (Compared to siControl for each foci, respectively). (B) BRCA1-deficient HCC1937 and complemented cell lines were treated with MMC (60 ng/ml) for 24h. The percentage of cells with > 5 foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to HCC1937+BRCA1 for each foci, respectively).

Mentions: Depletion of BRCA1 resulted in a strong reduction in FANCA, FANCG, FANCC, FANCL and FANCD2 foci formation in U2OS and HeLa cells (Fig 6A and S11A and S11B Fig), without affecting FANCA protein levels or FANCD2 monoubiquitination (S11C Fig). A similar reduction in FA core complex foci-containing cells was observed in BRCA1-deficient HCC1937, when compared to BRCA1-complemented HCC1937 (Fig 6B). To better characterize how BRCA1 promotes FA core complex foci formation, we then tested whether this function was mediated by known BRCA1-interacting proteins (S2 Table and S11D–S11H Fig). No defect in FANCA or FANCG foci was observed in cells deficient in FANCJ/BRIP1, FANCD1/BRCA2, FANCN/PALB2, FAM175A/ABRAXAS, UIMC1/RAP80 or RBBP8/CtIP, suggesting that BRCA1 performs this function independently, or through a binding partner other than the ones tested.


FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

Castella M, Jacquemont C, Thompson EL, Yeo JE, Cheung RS, Huang JW, Sobeck A, Hendrickson EA, Taniguchi T - PLoS Genet. (2015)

BRCA1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs, left untreated or treated with MMC (60 ng/ml) for 24h. The number of foci/cell (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05 (Compared to siControl for each foci, respectively). (B) BRCA1-deficient HCC1937 and complemented cell lines were treated with MMC (60 ng/ml) for 24h. The percentage of cells with > 5 foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to HCC1937+BRCA1 for each foci, respectively).
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Related In: Results  -  Collection

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pgen.1005563.g006: BRCA1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs, left untreated or treated with MMC (60 ng/ml) for 24h. The number of foci/cell (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05 (Compared to siControl for each foci, respectively). (B) BRCA1-deficient HCC1937 and complemented cell lines were treated with MMC (60 ng/ml) for 24h. The percentage of cells with > 5 foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to HCC1937+BRCA1 for each foci, respectively).
Mentions: Depletion of BRCA1 resulted in a strong reduction in FANCA, FANCG, FANCC, FANCL and FANCD2 foci formation in U2OS and HeLa cells (Fig 6A and S11A and S11B Fig), without affecting FANCA protein levels or FANCD2 monoubiquitination (S11C Fig). A similar reduction in FA core complex foci-containing cells was observed in BRCA1-deficient HCC1937, when compared to BRCA1-complemented HCC1937 (Fig 6B). To better characterize how BRCA1 promotes FA core complex foci formation, we then tested whether this function was mediated by known BRCA1-interacting proteins (S2 Table and S11D–S11H Fig). No defect in FANCA or FANCG foci was observed in cells deficient in FANCJ/BRIP1, FANCD1/BRCA2, FANCN/PALB2, FAM175A/ABRAXAS, UIMC1/RAP80 or RBBP8/CtIP, suggesting that BRCA1 performs this function independently, or through a binding partner other than the ones tested.

Bottom Line: Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle.ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment.Additionally, BRCA1 was required for efficient FA core complex foci formation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

No MeSH data available.


Related in: MedlinePlus