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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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Combinatorial IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Combinatorial Ku IP6-binding mutants had decreased IP6 binding relative to wild-type Ku. Filter binding assays were conducted as described in Materials and Methods section with 500 nM of recombinant Ku (wild-type or IP6-binding mutants) and 3H-IP6 (as indicated). Values shown represent the mean of at least two independent experiments with each measurement made in triplicate (N = minimum of 2 and n = minimum of 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM. (B) Increasing IP6 does not increase end joining in reactions complemented by combinatorial IP6-binding mutant Ku. Ku-depleted AS65 (20 µg) was complemented with 180 nM recombinant Ku (wild-type or IP6-binding mutants) and IP6 (as indicated), assayed for in vitro NHEJ, quantified as described in Materials and Methods section and normalized to the mean of end joining in the presence of wild-type Ku at 1 μM IP6. Values shown represent the mean of two independent experiments (N = 2 and n = minimum of 3). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment. (D) Comparison of end joining in reactions complemented with wild-type, single-subunit and combinatorial IP6-binding mutants of Ku. Reactions were carried out as described for (B). WT, wild-type Ku70/80; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80TM70DM, Ku70DM/Ku80TM.
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Figure 5: Combinatorial IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Combinatorial Ku IP6-binding mutants had decreased IP6 binding relative to wild-type Ku. Filter binding assays were conducted as described in Materials and Methods section with 500 nM of recombinant Ku (wild-type or IP6-binding mutants) and 3H-IP6 (as indicated). Values shown represent the mean of at least two independent experiments with each measurement made in triplicate (N = minimum of 2 and n = minimum of 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM. (B) Increasing IP6 does not increase end joining in reactions complemented by combinatorial IP6-binding mutant Ku. Ku-depleted AS65 (20 µg) was complemented with 180 nM recombinant Ku (wild-type or IP6-binding mutants) and IP6 (as indicated), assayed for in vitro NHEJ, quantified as described in Materials and Methods section and normalized to the mean of end joining in the presence of wild-type Ku at 1 μM IP6. Values shown represent the mean of two independent experiments (N = 2 and n = minimum of 3). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment. (D) Comparison of end joining in reactions complemented with wild-type, single-subunit and combinatorial IP6-binding mutants of Ku. Reactions were carried out as described for (B). WT, wild-type Ku70/80; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80TM70DM, Ku70DM/Ku80TM.

Mentions: When the combinatorial mutants were assayed for IP6 binding, we found that combining lysine-to-alanine mutations in Ku70 with those in Ku80 greatly reduced IP6 binding to 3.78% (Ku70DM/80DM) or 1.22% (Ku70DM/80TM) of that observed with wild-type Ku (Figure 5). While IP6 binding by the combinatorial IP6-binding mutants was severely diminished, the remaining IP6-binding activity was measurable and within the confidence limits of the IP6-binding assay. Therefore, the combinatorial mutants had significantly reduced, but not abolished, IP6-binding activity. The synergistic effects of combining Ku70DM with Ku80DM or Ku80TM further support the description of the Ku IP6-binding site as bipartite and demonstrate that we have correctly identified the IP6-binding site of Ku.Figure 5.


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)

Combinatorial IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Combinatorial Ku IP6-binding mutants had decreased IP6 binding relative to wild-type Ku. Filter binding assays were conducted as described in Materials and Methods section with 500 nM of recombinant Ku (wild-type or IP6-binding mutants) and 3H-IP6 (as indicated). Values shown represent the mean of at least two independent experiments with each measurement made in triplicate (N = minimum of 2 and n = minimum of 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM. (B) Increasing IP6 does not increase end joining in reactions complemented by combinatorial IP6-binding mutant Ku. Ku-depleted AS65 (20 µg) was complemented with 180 nM recombinant Ku (wild-type or IP6-binding mutants) and IP6 (as indicated), assayed for in vitro NHEJ, quantified as described in Materials and Methods section and normalized to the mean of end joining in the presence of wild-type Ku at 1 μM IP6. Values shown represent the mean of two independent experiments (N = 2 and n = minimum of 3). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment. (D) Comparison of end joining in reactions complemented with wild-type, single-subunit and combinatorial IP6-binding mutants of Ku. Reactions were carried out as described for (B). WT, wild-type Ku70/80; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80TM70DM, Ku70DM/Ku80TM.
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Figure 5: Combinatorial IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Combinatorial Ku IP6-binding mutants had decreased IP6 binding relative to wild-type Ku. Filter binding assays were conducted as described in Materials and Methods section with 500 nM of recombinant Ku (wild-type or IP6-binding mutants) and 3H-IP6 (as indicated). Values shown represent the mean of at least two independent experiments with each measurement made in triplicate (N = minimum of 2 and n = minimum of 6). Error bars show standard error. WT, wild-type Ku70/80; 80DM70DM, Ku70DM/Ku80DM; 80TM70DM, Ku70DM/Ku80TM. (B) Increasing IP6 does not increase end joining in reactions complemented by combinatorial IP6-binding mutant Ku. Ku-depleted AS65 (20 µg) was complemented with 180 nM recombinant Ku (wild-type or IP6-binding mutants) and IP6 (as indicated), assayed for in vitro NHEJ, quantified as described in Materials and Methods section and normalized to the mean of end joining in the presence of wild-type Ku at 1 μM IP6. Values shown represent the mean of two independent experiments (N = 2 and n = minimum of 3). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment. (D) Comparison of end joining in reactions complemented with wild-type, single-subunit and combinatorial IP6-binding mutants of Ku. Reactions were carried out as described for (B). WT, wild-type Ku70/80; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80; 80TM70DM, Ku70DM/Ku80TM.
Mentions: When the combinatorial mutants were assayed for IP6 binding, we found that combining lysine-to-alanine mutations in Ku70 with those in Ku80 greatly reduced IP6 binding to 3.78% (Ku70DM/80DM) or 1.22% (Ku70DM/80TM) of that observed with wild-type Ku (Figure 5). While IP6 binding by the combinatorial IP6-binding mutants was severely diminished, the remaining IP6-binding activity was measurable and within the confidence limits of the IP6-binding assay. Therefore, the combinatorial mutants had significantly reduced, but not abolished, IP6-binding activity. The synergistic effects of combining Ku70DM with Ku80DM or Ku80TM further support the description of the Ku IP6-binding site as bipartite and demonstrate that we have correctly identified the IP6-binding site of Ku.Figure 5.

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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