Limits...
Evidence for an inositol hexakisphosphate-dependent role for Ku in mammalian nonhomologous end joining that is independent of its role in the DNA-dependent protein kinase.

Cheung JC, Salerno B, Hanakahi LA - Nucleic Acids Res. (2008)

Bottom Line: Inositol hexakisphosphate (IP(6)) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP(6)-binding factor.Ku IP(6)-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku.Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP(6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA.

ABSTRACT
Nonhomologous end-joining (NHEJ) is an important pathway for the repair of DNA double-strand breaks (DSBs) and plays a critical role in maintaining genomic stability in mammalian cells. While Ku70/80 (Ku) functions in NHEJ as part of the DNA-dependent protein kinase (DNA-PK), genetic evidence indicates that the role of Ku in NHEJ goes beyond its participation in DNA-PK. Inositol hexakisphosphate (IP(6)) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP(6)-binding factor. Through mutational analysis, we identified a bipartite IP(6)-binding site in Ku and generated IP(6)-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP(6)-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP(6) binding and NHEJ. Ku IP(6)-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku. Our observations identify a hitherto undefined IP(6)-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro. Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP(6).

Show MeSH

Related in: MedlinePlus

Binding of IP6 by Ku. (A) Ku specifically binds to IP6, but not IS6. Filter binding assays were carried out as described in Materials and Methods section using 46.3 nM 3H-IP6 and 500 nM Ku in the presence of increasing amounts of unlabeled IP6 or IS6. Retained 3H-IP6 binding was measured as disintegration per minute (DPM). Values shown represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. (B) Identification of a putative IP6-binding site in Ku. The Ku70/80 ribbon structure is shown (PDB Accession No. 1JEY): Ku70 (red), Ku80 (yellow) and DNA (light gray). A cluster of conserved basic amino acids (blue space fill) is located near the Ku70/80 interface, away from the DNA-binding site and between the base of the Ku80 N-terminal α/β domain and the Ku70 central β-barrel domain. (C) Residues mutated in this study: 70DM—K357 K358 (pink space fill); 80DM—K238 K239 (dark gray space fill); and 80TM—K233 K238 K239 (K233 black space fill). Other basic amino acids that may be involved in Ku–IP6 interaction but were not examined in this study are shown in blue. Bottom view of (B), which is rotated 90°. (D) Expanded view of (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553570&req=5

Figure 2: Binding of IP6 by Ku. (A) Ku specifically binds to IP6, but not IS6. Filter binding assays were carried out as described in Materials and Methods section using 46.3 nM 3H-IP6 and 500 nM Ku in the presence of increasing amounts of unlabeled IP6 or IS6. Retained 3H-IP6 binding was measured as disintegration per minute (DPM). Values shown represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. (B) Identification of a putative IP6-binding site in Ku. The Ku70/80 ribbon structure is shown (PDB Accession No. 1JEY): Ku70 (red), Ku80 (yellow) and DNA (light gray). A cluster of conserved basic amino acids (blue space fill) is located near the Ku70/80 interface, away from the DNA-binding site and between the base of the Ku80 N-terminal α/β domain and the Ku70 central β-barrel domain. (C) Residues mutated in this study: 70DM—K357 K358 (pink space fill); 80DM—K238 K239 (dark gray space fill); and 80TM—K233 K238 K239 (K233 black space fill). Other basic amino acids that may be involved in Ku–IP6 interaction but were not examined in this study are shown in blue. Bottom view of (B), which is rotated 90°. (D) Expanded view of (C).

Mentions: 3H-IP6 was purchased from Perkin Elmer, Waltham, Massachusett (custom order). Analytical strong anion exchange (SAX) HPLC was used to determine that 80% of 3H counts were contained in 3H-IP6 and overall purity was 50% 3H-IP6. Attempts at preparative purification by SAX-HPLC were unsuccessful due to low specific activity and concentration of the 3H-IP6. Ku–3H-IP6 binding reactions (15 μl) were carried out in 20 mM HEPES pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1 mg/ml BSA, 40 mM KOAc, 10 mM DTT with 10 mM KPO4 pH 7.6 to provide an excess of phosphate to minimize nonspecific binding of 3H-IP6. The 46.3 nM 3H-IP6 and 500 nM Ku were found to produce maximum signal with minimum background. Reactions were incubated for 60 min. Nitrocellulose filter paper (Schleicher and Schuell, USA, BA85) was equilibrated with 20 mM HEPES pH 7.6, 0.5 mM EDTA, 10% glycerol, 10 mM KPO4 pH 8.0, and 1.5 mM IP6 after which the binding reaction was applied to the filter. Filters were washed 3 × with 20 mM HEPES, pH 7.6, 0.5 mM EDTA, 0.3 M NaCl then dried and 3H-IP6 was detected by scintillation counting. We determined that 100% of the Ku remained bound to the filter through this assay (data not shown). All solutions and steps were at 4°C. Competition assays (Figure 2A) were done by combining 3H-IP6 and unlabeled IP6 or inositol hexasulphate (IS6) before adding Ku to the reaction.Figure 2.


Evidence for an inositol hexakisphosphate-dependent role for Ku in mammalian nonhomologous end joining that is independent of its role in the DNA-dependent protein kinase.

Cheung JC, Salerno B, Hanakahi LA - Nucleic Acids Res. (2008)

Binding of IP6 by Ku. (A) Ku specifically binds to IP6, but not IS6. Filter binding assays were carried out as described in Materials and Methods section using 46.3 nM 3H-IP6 and 500 nM Ku in the presence of increasing amounts of unlabeled IP6 or IS6. Retained 3H-IP6 binding was measured as disintegration per minute (DPM). Values shown represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. (B) Identification of a putative IP6-binding site in Ku. The Ku70/80 ribbon structure is shown (PDB Accession No. 1JEY): Ku70 (red), Ku80 (yellow) and DNA (light gray). A cluster of conserved basic amino acids (blue space fill) is located near the Ku70/80 interface, away from the DNA-binding site and between the base of the Ku80 N-terminal α/β domain and the Ku70 central β-barrel domain. (C) Residues mutated in this study: 70DM—K357 K358 (pink space fill); 80DM—K238 K239 (dark gray space fill); and 80TM—K233 K238 K239 (K233 black space fill). Other basic amino acids that may be involved in Ku–IP6 interaction but were not examined in this study are shown in blue. Bottom view of (B), which is rotated 90°. (D) Expanded view of (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553570&req=5

Figure 2: Binding of IP6 by Ku. (A) Ku specifically binds to IP6, but not IS6. Filter binding assays were carried out as described in Materials and Methods section using 46.3 nM 3H-IP6 and 500 nM Ku in the presence of increasing amounts of unlabeled IP6 or IS6. Retained 3H-IP6 binding was measured as disintegration per minute (DPM). Values shown represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. (B) Identification of a putative IP6-binding site in Ku. The Ku70/80 ribbon structure is shown (PDB Accession No. 1JEY): Ku70 (red), Ku80 (yellow) and DNA (light gray). A cluster of conserved basic amino acids (blue space fill) is located near the Ku70/80 interface, away from the DNA-binding site and between the base of the Ku80 N-terminal α/β domain and the Ku70 central β-barrel domain. (C) Residues mutated in this study: 70DM—K357 K358 (pink space fill); 80DM—K238 K239 (dark gray space fill); and 80TM—K233 K238 K239 (K233 black space fill). Other basic amino acids that may be involved in Ku–IP6 interaction but were not examined in this study are shown in blue. Bottom view of (B), which is rotated 90°. (D) Expanded view of (C).
Mentions: 3H-IP6 was purchased from Perkin Elmer, Waltham, Massachusett (custom order). Analytical strong anion exchange (SAX) HPLC was used to determine that 80% of 3H counts were contained in 3H-IP6 and overall purity was 50% 3H-IP6. Attempts at preparative purification by SAX-HPLC were unsuccessful due to low specific activity and concentration of the 3H-IP6. Ku–3H-IP6 binding reactions (15 μl) were carried out in 20 mM HEPES pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1 mg/ml BSA, 40 mM KOAc, 10 mM DTT with 10 mM KPO4 pH 7.6 to provide an excess of phosphate to minimize nonspecific binding of 3H-IP6. The 46.3 nM 3H-IP6 and 500 nM Ku were found to produce maximum signal with minimum background. Reactions were incubated for 60 min. Nitrocellulose filter paper (Schleicher and Schuell, USA, BA85) was equilibrated with 20 mM HEPES pH 7.6, 0.5 mM EDTA, 10% glycerol, 10 mM KPO4 pH 8.0, and 1.5 mM IP6 after which the binding reaction was applied to the filter. Filters were washed 3 × with 20 mM HEPES, pH 7.6, 0.5 mM EDTA, 0.3 M NaCl then dried and 3H-IP6 was detected by scintillation counting. We determined that 100% of the Ku remained bound to the filter through this assay (data not shown). All solutions and steps were at 4°C. Competition assays (Figure 2A) were done by combining 3H-IP6 and unlabeled IP6 or inositol hexasulphate (IS6) before adding Ku to the reaction.Figure 2.

Bottom Line: Inositol hexakisphosphate (IP(6)) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP(6)-binding factor.Ku IP(6)-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku.Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP(6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA.

ABSTRACT
Nonhomologous end-joining (NHEJ) is an important pathway for the repair of DNA double-strand breaks (DSBs) and plays a critical role in maintaining genomic stability in mammalian cells. While Ku70/80 (Ku) functions in NHEJ as part of the DNA-dependent protein kinase (DNA-PK), genetic evidence indicates that the role of Ku in NHEJ goes beyond its participation in DNA-PK. Inositol hexakisphosphate (IP(6)) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP(6)-binding factor. Through mutational analysis, we identified a bipartite IP(6)-binding site in Ku and generated IP(6)-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP(6)-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP(6) binding and NHEJ. Ku IP(6)-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku. Our observations identify a hitherto undefined IP(6)-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro. Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP(6).

Show MeSH
Related in: MedlinePlus