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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

FA core complex foci form during S-G2 phases of the cell cycle.(A) U2OS cells were treated with MMC for 1h at 60ng/ml, then released into fresh media and fixed at the indicated time points. Cells were immunostained with the indicated antibodies and quantified. The percentage of cells with more than 5 foci is shown. (B) Cells were arrested in M-phase with nocodazole, re-seeded in fresh media and collected at the indicated times. The upper panel shows cell cycle profiles (PI staining). The lower panel shows percentage of cells with more than 5 FANCA foci (n = 3, mean ± SD). Cells were untreated or irradiated with 10Gy IR 1h before each time point. (C) Cells were irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-, FANCG- or FANCD2-foci containing cells population (n = 3, mean ± SD). Upper panel shows representative images. (D) Cells were transfected with indicated siRNAs and irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-foci containing cells (n = 3, mean ± SD).
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pgen.1005563.g002: FA core complex foci form during S-G2 phases of the cell cycle.(A) U2OS cells were treated with MMC for 1h at 60ng/ml, then released into fresh media and fixed at the indicated time points. Cells were immunostained with the indicated antibodies and quantified. The percentage of cells with more than 5 foci is shown. (B) Cells were arrested in M-phase with nocodazole, re-seeded in fresh media and collected at the indicated times. The upper panel shows cell cycle profiles (PI staining). The lower panel shows percentage of cells with more than 5 FANCA foci (n = 3, mean ± SD). Cells were untreated or irradiated with 10Gy IR 1h before each time point. (C) Cells were irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-, FANCG- or FANCD2-foci containing cells population (n = 3, mean ± SD). Upper panel shows representative images. (D) Cells were transfected with indicated siRNAs and irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-foci containing cells (n = 3, mean ± SD).

Mentions: The foci formation kinetics of FA core complex was similar to that of FANCD2, both after MMC pulse treatment (Fig 2A) and IR exposure (S5A Fig). Cell synchronization after release from nocodazole arrest revealed that FANCA foci were efficiently induced in S and G2 phases, in untreated cells or after IR, but not in G1 (Fig 2B). Furthermore, more than 95% of cells with FANCA or FANCG foci were cyclin A (a S/G2-phase marker)-positive (Fig 2C). These data indicate that FA core complex foci form during S and G2 phases. Similar results were obtained for FANCD2 foci (Fig 2C).


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)

FA core complex foci form during S-G2 phases of the cell cycle.(A) U2OS cells were treated with MMC for 1h at 60ng/ml, then released into fresh media and fixed at the indicated time points. Cells were immunostained with the indicated antibodies and quantified. The percentage of cells with more than 5 foci is shown. (B) Cells were arrested in M-phase with nocodazole, re-seeded in fresh media and collected at the indicated times. The upper panel shows cell cycle profiles (PI staining). The lower panel shows percentage of cells with more than 5 FANCA foci (n = 3, mean ± SD). Cells were untreated or irradiated with 10Gy IR 1h before each time point. (C) Cells were irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-, FANCG- or FANCD2-foci containing cells population (n = 3, mean ± SD). Upper panel shows representative images. (D) Cells were transfected with indicated siRNAs and irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-foci containing cells (n = 3, mean ± SD).
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005563.g002: FA core complex foci form during S-G2 phases of the cell cycle.(A) U2OS cells were treated with MMC for 1h at 60ng/ml, then released into fresh media and fixed at the indicated time points. Cells were immunostained with the indicated antibodies and quantified. The percentage of cells with more than 5 foci is shown. (B) Cells were arrested in M-phase with nocodazole, re-seeded in fresh media and collected at the indicated times. The upper panel shows cell cycle profiles (PI staining). The lower panel shows percentage of cells with more than 5 FANCA foci (n = 3, mean ± SD). Cells were untreated or irradiated with 10Gy IR 1h before each time point. (C) Cells were irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-, FANCG- or FANCD2-foci containing cells population (n = 3, mean ± SD). Upper panel shows representative images. (D) Cells were transfected with indicated siRNAs and irradiated with 10Gy IR 2h before fixation. Graph shows percentage of cyclin A-positive and -negative cells in the FANCA-foci containing cells (n = 3, mean ± SD).
Mentions: The foci formation kinetics of FA core complex was similar to that of FANCD2, both after MMC pulse treatment (Fig 2A) and IR exposure (S5A Fig). Cell synchronization after release from nocodazole arrest revealed that FANCA foci were efficiently induced in S and G2 phases, in untreated cells or after IR, but not in G1 (Fig 2B). Furthermore, more than 95% of cells with FANCA or FANCG foci were cyclin A (a S/G2-phase marker)-positive (Fig 2C). These data indicate that FA core complex foci form during S and G2 phases. Similar results were obtained for FANCD2 foci (Fig 2C).

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