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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

Deubiquitination of FANCI by USP1 is required for FA core complex foci formation.(A) FANCI-deficient F010191 cells were transfected with the indicated siRNAs, treated with MMC and then fixed and stained with FANCA antibody. Percentage of cells containing 5 or more foci is shown (n = 3, mean ± SD). (n.s.) indicates no statistical significance. Protein extracts were subjected to western blotting to confirm depletion of USP1 (lower panel). (B) U2OS cells transduced with the indicated constructs were transfected with the siControl or siUSP1. 48h later they were treated with MMC and then fixed and stained with FANCA antibody. Upper panel shows percentage of cells containing 5 or more foci (n = 3, mean ± SD). (*) Indicates p<0.05. (n.s.) indicates no statistical significance. Lower panel shows protein extracts from the same experiment subjected to western blotting.
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pgen.1005563.g008: Deubiquitination of FANCI by USP1 is required for FA core complex foci formation.(A) FANCI-deficient F010191 cells were transfected with the indicated siRNAs, treated with MMC and then fixed and stained with FANCA antibody. Percentage of cells containing 5 or more foci is shown (n = 3, mean ± SD). (n.s.) indicates no statistical significance. Protein extracts were subjected to western blotting to confirm depletion of USP1 (lower panel). (B) U2OS cells transduced with the indicated constructs were transfected with the siControl or siUSP1. 48h later they were treated with MMC and then fixed and stained with FANCA antibody. Upper panel shows percentage of cells containing 5 or more foci (n = 3, mean ± SD). (*) Indicates p<0.05. (n.s.) indicates no statistical significance. Lower panel shows protein extracts from the same experiment subjected to western blotting.

Mentions: Since both non-ubiquitinated FANCI and USP1 catalytic activity promoted FA core complex foci formation, next we tested the possibility that FANCI was the relevant substrate for USP1 in this function. We first tested epistasis between FANCI and USP1. USP1 was depleted using siRNA from FANCI-deficient F010191 cells. As shown in Fig 8A, USP1 depletion did not result in an increased loss of FANCA foci in FANCI-deficient cells, suggesting that FANCI and USP1 promote FA core complex foci formation through the same mechanism. If FANCI is the relevant USP1 substrate to promote FA core complex formation, overexpression of a non-ubiquitinatable FANCI should be able to rescue FA core complex foci formation in USP1-depleted cells. Therefore, we overexpressed wild-type or K523R mutant of FANCI in cells that were either transfected with siRNA control or siUSP1. As shown in Fig 8B, overexpression of the FANCI K523R mutant partially rescued FANCA foci formation in USP1-depleted cells, while the wild-type FANCI was not able to do so. These results suggest that FANCI needs to be deubiquitinated by USP1 to promote FA core complex foci efficiently.


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)

Deubiquitination of FANCI by USP1 is required for FA core complex foci formation.(A) FANCI-deficient F010191 cells were transfected with the indicated siRNAs, treated with MMC and then fixed and stained with FANCA antibody. Percentage of cells containing 5 or more foci is shown (n = 3, mean ± SD). (n.s.) indicates no statistical significance. Protein extracts were subjected to western blotting to confirm depletion of USP1 (lower panel). (B) U2OS cells transduced with the indicated constructs were transfected with the siControl or siUSP1. 48h later they were treated with MMC and then fixed and stained with FANCA antibody. Upper panel shows percentage of cells containing 5 or more foci (n = 3, mean ± SD). (*) Indicates p<0.05. (n.s.) indicates no statistical significance. Lower panel shows protein extracts from the same experiment subjected to western blotting.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pgen.1005563.g008: Deubiquitination of FANCI by USP1 is required for FA core complex foci formation.(A) FANCI-deficient F010191 cells were transfected with the indicated siRNAs, treated with MMC and then fixed and stained with FANCA antibody. Percentage of cells containing 5 or more foci is shown (n = 3, mean ± SD). (n.s.) indicates no statistical significance. Protein extracts were subjected to western blotting to confirm depletion of USP1 (lower panel). (B) U2OS cells transduced with the indicated constructs were transfected with the siControl or siUSP1. 48h later they were treated with MMC and then fixed and stained with FANCA antibody. Upper panel shows percentage of cells containing 5 or more foci (n = 3, mean ± SD). (*) Indicates p<0.05. (n.s.) indicates no statistical significance. Lower panel shows protein extracts from the same experiment subjected to western blotting.
Mentions: Since both non-ubiquitinated FANCI and USP1 catalytic activity promoted FA core complex foci formation, next we tested the possibility that FANCI was the relevant substrate for USP1 in this function. We first tested epistasis between FANCI and USP1. USP1 was depleted using siRNA from FANCI-deficient F010191 cells. As shown in Fig 8A, USP1 depletion did not result in an increased loss of FANCA foci in FANCI-deficient cells, suggesting that FANCI and USP1 promote FA core complex foci formation through the same mechanism. If FANCI is the relevant USP1 substrate to promote FA core complex formation, overexpression of a non-ubiquitinatable FANCI should be able to rescue FA core complex foci formation in USP1-depleted cells. Therefore, we overexpressed wild-type or K523R mutant of FANCI in cells that were either transfected with siRNA control or siUSP1. As shown in Fig 8B, overexpression of the FANCI K523R mutant partially rescued FANCA foci formation in USP1-depleted cells, while the wild-type FANCI was not able to do so. These results suggest that FANCI needs to be deubiquitinated by USP1 to promote FA core complex foci efficiently.

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