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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

ATR is required for FA core complex foci formation.(A) ATR deficient F02-98tert cells and complemented cells were treated with 60ng/ml MMC for 24h before fixation and staining. Percentage of cells with >5 foci (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance. The left panel shows ATR protein expression in these cell lines (western blotting). (B) U2OS cells were pretreated with 10μM VE-821 for 2h and then treated with MMC (60ng/ml) for 24h in the presence of VE-821. Then, cells were fixed and immunostained with the indicated antibodies. Percentage of cells with >5 foci is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to “MMC treated—no VE-821” sample). (C) Cells were treated with the same conditions as in (B). pCHK1 (Serine 345) western blotting confirmed ATR inhibition by VE-821.
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pgen.1005563.g003: ATR is required for FA core complex foci formation.(A) ATR deficient F02-98tert cells and complemented cells were treated with 60ng/ml MMC for 24h before fixation and staining. Percentage of cells with >5 foci (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance. The left panel shows ATR protein expression in these cell lines (western blotting). (B) U2OS cells were pretreated with 10μM VE-821 for 2h and then treated with MMC (60ng/ml) for 24h in the presence of VE-821. Then, cells were fixed and immunostained with the indicated antibodies. Percentage of cells with >5 foci is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to “MMC treated—no VE-821” sample). (C) Cells were treated with the same conditions as in (B). pCHK1 (Serine 345) western blotting confirmed ATR inhibition by VE-821.

Mentions: The ability to detect recruitment of FA core complex proteins at sites of DNA damage allowed us to search for factors required for the formation of these foci and therefore gain deeper insight into how the FA pathway is regulated. ATR is the primary kinase that controls FA pathway activation [23, 24, 33]. However, whether ATR is required for FA core complex recruitment is unknown. A strong reduction in the number of cells containing FANCA, FANCG, FANCC, FANCL or FANCD2 foci was observed in the ATR-deficient cells (F02-98 fibroblasts and ATR-depleted U2OS cells), but not in ATR-proficient control cells (Fig 3A and S6A and S6B Fig). ATR specific inhibitor (VE-821) [34], but not ATM inhibitor, impaired the formation of both FA core complex and FANCD2 foci (Fig 3B and S3C, S6C and S6D Fig), indicating that ATR kinase activity is required for FA core complex foci formation.


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)

ATR is required for FA core complex foci formation.(A) ATR deficient F02-98tert cells and complemented cells were treated with 60ng/ml MMC for 24h before fixation and staining. Percentage of cells with >5 foci (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance. The left panel shows ATR protein expression in these cell lines (western blotting). (B) U2OS cells were pretreated with 10μM VE-821 for 2h and then treated with MMC (60ng/ml) for 24h in the presence of VE-821. Then, cells were fixed and immunostained with the indicated antibodies. Percentage of cells with >5 foci is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to “MMC treated—no VE-821” sample). (C) Cells were treated with the same conditions as in (B). pCHK1 (Serine 345) western blotting confirmed ATR inhibition by VE-821.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4592014&req=5

pgen.1005563.g003: ATR is required for FA core complex foci formation.(A) ATR deficient F02-98tert cells and complemented cells were treated with 60ng/ml MMC for 24h before fixation and staining. Percentage of cells with >5 foci (n = 3, mean ± SD) and representative images are shown. (*) Indicates p<0.05; (n.s.) indicates no statistical significance. The left panel shows ATR protein expression in these cell lines (western blotting). (B) U2OS cells were pretreated with 10μM VE-821 for 2h and then treated with MMC (60ng/ml) for 24h in the presence of VE-821. Then, cells were fixed and immunostained with the indicated antibodies. Percentage of cells with >5 foci is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (n.s.) indicates no statistical significance (Compared to “MMC treated—no VE-821” sample). (C) Cells were treated with the same conditions as in (B). pCHK1 (Serine 345) western blotting confirmed ATR inhibition by VE-821.
Mentions: The ability to detect recruitment of FA core complex proteins at sites of DNA damage allowed us to search for factors required for the formation of these foci and therefore gain deeper insight into how the FA pathway is regulated. ATR is the primary kinase that controls FA pathway activation [23, 24, 33]. However, whether ATR is required for FA core complex recruitment is unknown. A strong reduction in the number of cells containing FANCA, FANCG, FANCC, FANCL or FANCD2 foci was observed in the ATR-deficient cells (F02-98 fibroblasts and ATR-depleted U2OS cells), but not in ATR-proficient control cells (Fig 3A and S6A and S6B Fig). ATR specific inhibitor (VE-821) [34], but not ATM inhibitor, impaired the formation of both FA core complex and FANCD2 foci (Fig 3B and S3C, S6C and S6D Fig), indicating that ATR kinase activity is required for FA core complex foci formation.

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