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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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Characterization of single-subunit IP6-binding mutants of Ku. (A) Purified Ku proteins used in this study. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80. Mutant heterodimers were produced by coexpressing K-to-A mutant subunits with the corresponding wild-type subunit followed by purification as described in Materials and Methods section. A total of 0.5 µg total protein was resolved on 8% SDS–PAGE and silver stained. (B) Single-subunit 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; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80.
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Figure 3: Characterization of single-subunit IP6-binding mutants of Ku. (A) Purified Ku proteins used in this study. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80. Mutant heterodimers were produced by coexpressing K-to-A mutant subunits with the corresponding wild-type subunit followed by purification as described in Materials and Methods section. A total of 0.5 µg total protein was resolved on 8% SDS–PAGE and silver stained. (B) Single-subunit 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; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80.

Mentions: When the Ku single subunit lysine-to-alanine mutants were assayed for IP6 binding we found that, for all three of the single-subunit mutant heterodimers tested, IP6-binding was reduced relative to binding by wild-type Ku (Figure 3B). Limitations associated with the 3H-IP6 (see Materials and Methods section) prevented binding measurements at concentrations >500 nM, making binding constant measurements imprecise and restricting this analysis to relative binding by the recombinant Ku proteins.


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)

Characterization of single-subunit IP6-binding mutants of Ku. (A) Purified Ku proteins used in this study. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80. Mutant heterodimers were produced by coexpressing K-to-A mutant subunits with the corresponding wild-type subunit followed by purification as described in Materials and Methods section. A total of 0.5 µg total protein was resolved on 8% SDS–PAGE and silver stained. (B) Single-subunit 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; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80.
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Figure 3: Characterization of single-subunit IP6-binding mutants of Ku. (A) Purified Ku proteins used in this study. WT, wild-type Ku70/80; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80. Mutant heterodimers were produced by coexpressing K-to-A mutant subunits with the corresponding wild-type subunit followed by purification as described in Materials and Methods section. A total of 0.5 µg total protein was resolved on 8% SDS–PAGE and silver stained. (B) Single-subunit 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; 80DM, Ku70/Ku80DM; 80TM, Ku70/Ku80TM; 70DM, Ku70DM/Ku80.
Mentions: When the Ku single subunit lysine-to-alanine mutants were assayed for IP6 binding we found that, for all three of the single-subunit mutant heterodimers tested, IP6-binding was reduced relative to binding by wild-type Ku (Figure 3B). Limitations associated with the 3H-IP6 (see Materials and Methods section) prevented binding measurements at concentrations >500 nM, making binding constant measurements imprecise and restricting this analysis to relative binding by the recombinant Ku proteins.

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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