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PMID:23324393

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Citation

Postow, L and Funabiki, H (2013) An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation. Cell Cycle 12:587-95

Abstract

The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.

Links

PubMed PMC3594259 Online version:10.4161/cc.23408

Keywords

Animals; Antigens, Nuclear/genetics; Antigens, Nuclear/metabolism; Cell-Free System; Cullin Proteins/genetics; Cullin Proteins/metabolism; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; F-Box Proteins/genetics; F-Box Proteins/metabolism; Gene Expression Regulation; Molecular Sequence Data; Protein Binding; Protein Multimerization; S-Phase Kinase-Associated Proteins/genetics; S-Phase Kinase-Associated Proteins/metabolism; Sequence Alignment; Signal Transduction; Ubiquitination; Xenopus Proteins/genetics; Xenopus Proteins/metabolism; Xenopus laevis/genetics; Xenopus laevis/growth & development; Xenopus laevis/metabolism; Zygote/chemistry

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

XENTR:A9JSE6

GO:0003677: DNA binding

ECO:0000314:

F

See Table 1 and Figure 2.

complete
CACAO 9906

XENLA:Q63ZQ7

GO:2001032: regulation of double-strand break repair via nonhomologous end joining

ECO:0000314:

P

Figure 1 shows that the SCF complex (Cul1 and Skp1) is required for Ku80 ubiquitylation. Ku80 is the initiating factor of nonhomologous end joining double-strand break repair. When polyubiquitylated, Ku80 dissociates from the DNA where the break was. Figure 4 (an immunoprecipitation) shows that when Fbxl2 is not present, the binding of Cul1 and Skp1 is inhibited and there are less modifications to Ku80. This proves that Fbx12 is the protein that recruits the SCF to double stranded breaks.

complete
CACAO 9796

XENLA:Q63ZQ7

GO:0003677: DNA binding

ECO:0000314:

F

See Table 1 and Figure 2.

complete
CACAO 9907

XENLA:Q63ZQ7

involved_in

GO:2001032: regulation of double-strand break repair via nonhomologous end joining

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

See also

References

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