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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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Single-subunit IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Top: Ku-depleted AS65 (20 µg) was complemented with 1 μM IP6 and assayed for in vitro NHEJ as described in Materials and Methods section in the absence (lane 3) or presence (lanes 4–7) of recombinant Ku (180 nM, wild-type or IP6-binding mutants). End joining observed in the absence of Ku (lane 3) was not sensitive to neutralizing anti-XRCC4 antibodies (Supplementary Figure 3A) and does not represent background levels of NHEJ. Mock-depleted AS65, anti-Ku antibodies omitted from immunodepletion. The negative sign indicates no protein. Top panel was assembled from lanes from a single autoradiogram. Bottom: anti-Ku70 Western blot shows the amount of endogenous Ku70 (lanes 2, 3) or recombinant Ku70 (lanes 4–7) present in the end-joining reactions (Top). Lane 2 shows the amount of endogenous Ku present in mock-depleted AS65 and lane 3 shows the extent of Ku depletion. Bottom panel was assembled from lanes from a single western blot. (B) Increasing IP6 increases end joining in reactions complemented by single-subunit IP6-binding mutants of 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 with each measurement made in duplicate (N = 2 and n = 4). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment.
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Figure 4: Single-subunit IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Top: Ku-depleted AS65 (20 µg) was complemented with 1 μM IP6 and assayed for in vitro NHEJ as described in Materials and Methods section in the absence (lane 3) or presence (lanes 4–7) of recombinant Ku (180 nM, wild-type or IP6-binding mutants). End joining observed in the absence of Ku (lane 3) was not sensitive to neutralizing anti-XRCC4 antibodies (Supplementary Figure 3A) and does not represent background levels of NHEJ. Mock-depleted AS65, anti-Ku antibodies omitted from immunodepletion. The negative sign indicates no protein. Top panel was assembled from lanes from a single autoradiogram. Bottom: anti-Ku70 Western blot shows the amount of endogenous Ku70 (lanes 2, 3) or recombinant Ku70 (lanes 4–7) present in the end-joining reactions (Top). Lane 2 shows the amount of endogenous Ku present in mock-depleted AS65 and lane 3 shows the extent of Ku depletion. Bottom panel was assembled from lanes from a single western blot. (B) Increasing IP6 increases end joining in reactions complemented by single-subunit IP6-binding mutants of 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 with each measurement made in duplicate (N = 2 and n = 4). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment.

Mentions: To assess the ability of Ku IP6-binding mutants to participate in NHEJ, we developed an assay system that required addition of both Ku and IP6 for NHEJ in vitro. Starting with the AS65 fraction, which required addition of IP6 for efficient NHEJ, high-salt conditions were used to disrupt Ku–protein and Ku–DNA interactions and selectively immunodepleted the abundant Ku protein (12). We confirmed that the Ku-depleted extracts contained no detectable levels of Ku70 or Ku80, but that the levels of other NHEJ factors were unaffected (Supplementary Figure 1). In the presence of IP6, Ku-depleted AS65 had reduced end-joining activity relative to mock-depleted AS65, which was consistent with depletion of Ku (Figure 4A and Supplementary Figure 3). The end joining that was observed in the Ku-depleted AS65 was not sensitive to treatment with neutralizing anti-XRCC4 antibodies (Supplementary Figure 3A). We attribute this Ku-, XRCC4-independent concatamer formation to DNA ligases I and III, which had increased access to the DNA ends in the absence of Ku and were present in the Ku-depleted AS65 (Supplementary Figure 1). We conclude that the end joining observed in the Ku-depleted AS65 (Figure 4A, lane 3) does not represent background levels of ligase IV-mediated NHEJ, but rather adventitious concatamer formation by ligase I or III.Figure 4.


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)

Single-subunit IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Top: Ku-depleted AS65 (20 µg) was complemented with 1 μM IP6 and assayed for in vitro NHEJ as described in Materials and Methods section in the absence (lane 3) or presence (lanes 4–7) of recombinant Ku (180 nM, wild-type or IP6-binding mutants). End joining observed in the absence of Ku (lane 3) was not sensitive to neutralizing anti-XRCC4 antibodies (Supplementary Figure 3A) and does not represent background levels of NHEJ. Mock-depleted AS65, anti-Ku antibodies omitted from immunodepletion. The negative sign indicates no protein. Top panel was assembled from lanes from a single autoradiogram. Bottom: anti-Ku70 Western blot shows the amount of endogenous Ku70 (lanes 2, 3) or recombinant Ku70 (lanes 4–7) present in the end-joining reactions (Top). Lane 2 shows the amount of endogenous Ku present in mock-depleted AS65 and lane 3 shows the extent of Ku depletion. Bottom panel was assembled from lanes from a single western blot. (B) Increasing IP6 increases end joining in reactions complemented by single-subunit IP6-binding mutants of 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 with each measurement made in duplicate (N = 2 and n = 4). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Single-subunit IP6-binding mutants of Ku are impaired for complementation of end joining in vitro. (A) Top: Ku-depleted AS65 (20 µg) was complemented with 1 μM IP6 and assayed for in vitro NHEJ as described in Materials and Methods section in the absence (lane 3) or presence (lanes 4–7) of recombinant Ku (180 nM, wild-type or IP6-binding mutants). End joining observed in the absence of Ku (lane 3) was not sensitive to neutralizing anti-XRCC4 antibodies (Supplementary Figure 3A) and does not represent background levels of NHEJ. Mock-depleted AS65, anti-Ku antibodies omitted from immunodepletion. The negative sign indicates no protein. Top panel was assembled from lanes from a single autoradiogram. Bottom: anti-Ku70 Western blot shows the amount of endogenous Ku70 (lanes 2, 3) or recombinant Ku70 (lanes 4–7) present in the end-joining reactions (Top). Lane 2 shows the amount of endogenous Ku present in mock-depleted AS65 and lane 3 shows the extent of Ku depletion. Bottom panel was assembled from lanes from a single western blot. (B) Increasing IP6 increases end joining in reactions complemented by single-subunit IP6-binding mutants of 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 with each measurement made in duplicate (N = 2 and n = 4). (C) Representative FLA-7000 Image Reader images used to generate (B). Figure was assembled from results from a single experiment.
Mentions: To assess the ability of Ku IP6-binding mutants to participate in NHEJ, we developed an assay system that required addition of both Ku and IP6 for NHEJ in vitro. Starting with the AS65 fraction, which required addition of IP6 for efficient NHEJ, high-salt conditions were used to disrupt Ku–protein and Ku–DNA interactions and selectively immunodepleted the abundant Ku protein (12). We confirmed that the Ku-depleted extracts contained no detectable levels of Ku70 or Ku80, but that the levels of other NHEJ factors were unaffected (Supplementary Figure 1). In the presence of IP6, Ku-depleted AS65 had reduced end-joining activity relative to mock-depleted AS65, which was consistent with depletion of Ku (Figure 4A and Supplementary Figure 3). The end joining that was observed in the Ku-depleted AS65 was not sensitive to treatment with neutralizing anti-XRCC4 antibodies (Supplementary Figure 3A). We attribute this Ku-, XRCC4-independent concatamer formation to DNA ligases I and III, which had increased access to the DNA ends in the absence of Ku and were present in the Ku-depleted AS65 (Supplementary Figure 1). We conclude that the end joining observed in the Ku-depleted AS65 (Figure 4A, lane 3) does not represent background levels of ligase IV-mediated NHEJ, but rather adventitious concatamer formation by ligase I or III.Figure 4.

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