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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).

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Separation of IP6-binding- and DNA-PK-related functions of Ku. (A) dsDNA binding by IP6-binding mutants of Ku. EMSA carried out with 5 nM of 32P-end labeled dsDNA and recombinant Ku (as indicated) at 0, 2.5, 5 and 10 nM. Complexes were resolved on 5% native PAGE. Free dsDNA and dsDNA bound by 1 Ku protein as indicated. (B) DNA binding by wild-type and IP6-binding mutants of Ku. EMSA was carried out as described for (A) with recombinant Ku proteins as indicated and quantified as described in Materials and Methods section and expressed as percentage of protein-bound DNA. Values shown represent the mean of three independent experiments. (C) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of single-subunit IP6-binding mutants. DNA-PK assays were carried out as described using Ku-free DNA-PKcs (0.463 pmol) purified from HeLa cells and 0, 0.25, 0.6, 0.9 or 1.5 pmol recombinant Ku (wild-type or IP6-binding mutants) and 10 µg/ml DNA. (D) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of combinatorial IP6-binding mutants. DNA-PK assays were carried out as described for (B) with recombinant Ku proteins as indicated. Values shown for DNA-PK assays represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM.
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Figure 6: Separation of IP6-binding- and DNA-PK-related functions of Ku. (A) dsDNA binding by IP6-binding mutants of Ku. EMSA carried out with 5 nM of 32P-end labeled dsDNA and recombinant Ku (as indicated) at 0, 2.5, 5 and 10 nM. Complexes were resolved on 5% native PAGE. Free dsDNA and dsDNA bound by 1 Ku protein as indicated. (B) DNA binding by wild-type and IP6-binding mutants of Ku. EMSA was carried out as described for (A) with recombinant Ku proteins as indicated and quantified as described in Materials and Methods section and expressed as percentage of protein-bound DNA. Values shown represent the mean of three independent experiments. (C) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of single-subunit IP6-binding mutants. DNA-PK assays were carried out as described using Ku-free DNA-PKcs (0.463 pmol) purified from HeLa cells and 0, 0.25, 0.6, 0.9 or 1.5 pmol recombinant Ku (wild-type or IP6-binding mutants) and 10 µg/ml DNA. (D) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of combinatorial IP6-binding mutants. DNA-PK assays were carried out as described for (B) with recombinant Ku proteins as indicated. Values shown for DNA-PK assays represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM.

Mentions: First, we compared DNA binding by wild-type recombinant Ku, the single-subunit mutants Ku70/80TM and Ku70DM/80 and the combinatorial mutants Ku70DM/80DM and Ku70DM/80TM. The DNA used in this study, originally described by Walker et al. (8), uses a short piece of duplex DNA that can accommodate only one Ku protein. The termini of the synthetic duplex DNA permit directional loading of Ku from only one exposed end. Loading from the second terminus is prevented by the presence of an unpaired region, which can form a hammerhead structure (8). We used EMSA to assess DNA binding by wild-type and IP6-binding mutants of Ku. A representative EMSA (Figure 6A) and the cumulative results of three experiments (Figure 6B) show that binding of a single Ku protein to DNA is comparable for all of the Ku proteins tested.Figure 6.


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)

Separation of IP6-binding- and DNA-PK-related functions of Ku. (A) dsDNA binding by IP6-binding mutants of Ku. EMSA carried out with 5 nM of 32P-end labeled dsDNA and recombinant Ku (as indicated) at 0, 2.5, 5 and 10 nM. Complexes were resolved on 5% native PAGE. Free dsDNA and dsDNA bound by 1 Ku protein as indicated. (B) DNA binding by wild-type and IP6-binding mutants of Ku. EMSA was carried out as described for (A) with recombinant Ku proteins as indicated and quantified as described in Materials and Methods section and expressed as percentage of protein-bound DNA. Values shown represent the mean of three independent experiments. (C) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of single-subunit IP6-binding mutants. DNA-PK assays were carried out as described using Ku-free DNA-PKcs (0.463 pmol) purified from HeLa cells and 0, 0.25, 0.6, 0.9 or 1.5 pmol recombinant Ku (wild-type or IP6-binding mutants) and 10 µg/ml DNA. (D) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of combinatorial IP6-binding mutants. DNA-PK assays were carried out as described for (B) with recombinant Ku proteins as indicated. Values shown for DNA-PK assays represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM.
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Figure 6: Separation of IP6-binding- and DNA-PK-related functions of Ku. (A) dsDNA binding by IP6-binding mutants of Ku. EMSA carried out with 5 nM of 32P-end labeled dsDNA and recombinant Ku (as indicated) at 0, 2.5, 5 and 10 nM. Complexes were resolved on 5% native PAGE. Free dsDNA and dsDNA bound by 1 Ku protein as indicated. (B) DNA binding by wild-type and IP6-binding mutants of Ku. EMSA was carried out as described for (A) with recombinant Ku proteins as indicated and quantified as described in Materials and Methods section and expressed as percentage of protein-bound DNA. Values shown represent the mean of three independent experiments. (C) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of single-subunit IP6-binding mutants. DNA-PK assays were carried out as described using Ku-free DNA-PKcs (0.463 pmol) purified from HeLa cells and 0, 0.25, 0.6, 0.9 or 1.5 pmol recombinant Ku (wild-type or IP6-binding mutants) and 10 µg/ml DNA. (D) Mutation of the IP6-binding site did not affect activation of DNA-PKcs—analysis of combinatorial IP6-binding mutants. DNA-PK assays were carried out as described for (B) with recombinant Ku proteins as indicated. Values shown for DNA-PK assays represent the mean of two independent experiments with each measurement made in triplicate (N = 2 and n = 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM.
Mentions: First, we compared DNA binding by wild-type recombinant Ku, the single-subunit mutants Ku70/80TM and Ku70DM/80 and the combinatorial mutants Ku70DM/80DM and Ku70DM/80TM. The DNA used in this study, originally described by Walker et al. (8), uses a short piece of duplex DNA that can accommodate only one Ku protein. The termini of the synthetic duplex DNA permit directional loading of Ku from only one exposed end. Loading from the second terminus is prevented by the presence of an unpaired region, which can form a hammerhead structure (8). We used EMSA to assess DNA binding by wild-type and IP6-binding mutants of Ku. A representative EMSA (Figure 6A) and the cumulative results of three experiments (Figure 6B) show that binding of a single Ku protein to DNA is comparable for all of the Ku proteins tested.Figure 6.

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