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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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IP6 is required for NHEJ in vitro. (A) Structure of IP6. With d-myo-inositol as its base ring structure, IP6 has one axial phosphate group (C-2), five phosphate groups in equatorial position and a molecular weight of 660 Da. (B) End-joining assays were carried out using 20 µg of HeLa WCE or AS65 as described in Materials and Methods section and complemented with IP6 or IS6. Treatment with Wortmannin (3 μM, lanes 4 and 10) and anti-XRCC4 antibodies (α-XRCC4, 1:250 dilution, lanes 3 and 9) demonstrate that the observed end joining is bona fide NHEJ. A −ve sign indicates no protein. Monomer, monomeric DNA substrate; dimer, trimer, etc., end-joined concatamers. (C) IP6 increased the rate of NHEJ in vitro. AS65 (20 µg) was assayed for end joining as described in Materials and Methods section in the presence (left) or absence (right) of 1 μM IP6 for the indicated time. (D) Quantification of C presented as percent of ends joined as a function of reaction time. Graph includes controls in which reactions were treated with anti-XRCC4 antibodies (1:250 dilution) to inhibit NHEJ (filled triangle, + IP6 + α-XRCC4; open triangle, − IP6 + α-XRCC4), which are not shown in (C).
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Figure 1: IP6 is required for NHEJ in vitro. (A) Structure of IP6. With d-myo-inositol as its base ring structure, IP6 has one axial phosphate group (C-2), five phosphate groups in equatorial position and a molecular weight of 660 Da. (B) End-joining assays were carried out using 20 µg of HeLa WCE or AS65 as described in Materials and Methods section and complemented with IP6 or IS6. Treatment with Wortmannin (3 μM, lanes 4 and 10) and anti-XRCC4 antibodies (α-XRCC4, 1:250 dilution, lanes 3 and 9) demonstrate that the observed end joining is bona fide NHEJ. A −ve sign indicates no protein. Monomer, monomeric DNA substrate; dimer, trimer, etc., end-joined concatamers. (C) IP6 increased the rate of NHEJ in vitro. AS65 (20 µg) was assayed for end joining as described in Materials and Methods section in the presence (left) or absence (right) of 1 μM IP6 for the indicated time. (D) Quantification of C presented as percent of ends joined as a function of reaction time. Graph includes controls in which reactions were treated with anti-XRCC4 antibodies (1:250 dilution) to inhibit NHEJ (filled triangle, + IP6 + α-XRCC4; open triangle, − IP6 + α-XRCC4), which are not shown in (C).

Mentions: We have shown that, in addition to activating DNA-PKcs, Ku binds the small, phosphate-rich molecule inositol hexakisphosphate (IP6, Figure 1A), which we previously identified as a stimulatory factor in mammalian NHEJ (9,10). Subsequent publications demonstrated that binding of IP6 by Ku did not change the ability of Ku to bind DNA or to assemble with DNA-PKcs to form DNA-PK (10,11). These data suggest that the role of IP6 in mammalian NHEJ is tied to functions of Ku that are independent of DNA-PK. To determine if binding of IP6 by Ku contributes to efficient NHEJ, we used mutational analysis to identify an IP6-binding site in Ku. This mutational analysis revealed that the Ku IP6-binding site, like its DNA-binding site, is bipartite and requires residues from both Ku70 and Ku80 subunits. IP6-binding mutants of Ku were separation-of-function mutants that stimulated DNA-PK but showed reduced affinity for IP6. These separation-of-function mutants were also impaired for complementation of NHEJ in Ku-depleted extracts, which demonstrates that formation of a Ku–IP6 complex is important for efficient NHEJ in vitro. These data show that the role of Ku in mammalian NHEJ may be divided into DNA-PKcs-dependent and IP6-dependent functions.Figure 1.


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)

IP6 is required for NHEJ in vitro. (A) Structure of IP6. With d-myo-inositol as its base ring structure, IP6 has one axial phosphate group (C-2), five phosphate groups in equatorial position and a molecular weight of 660 Da. (B) End-joining assays were carried out using 20 µg of HeLa WCE or AS65 as described in Materials and Methods section and complemented with IP6 or IS6. Treatment with Wortmannin (3 μM, lanes 4 and 10) and anti-XRCC4 antibodies (α-XRCC4, 1:250 dilution, lanes 3 and 9) demonstrate that the observed end joining is bona fide NHEJ. A −ve sign indicates no protein. Monomer, monomeric DNA substrate; dimer, trimer, etc., end-joined concatamers. (C) IP6 increased the rate of NHEJ in vitro. AS65 (20 µg) was assayed for end joining as described in Materials and Methods section in the presence (left) or absence (right) of 1 μM IP6 for the indicated time. (D) Quantification of C presented as percent of ends joined as a function of reaction time. Graph includes controls in which reactions were treated with anti-XRCC4 antibodies (1:250 dilution) to inhibit NHEJ (filled triangle, + IP6 + α-XRCC4; open triangle, − IP6 + α-XRCC4), which are not shown in (C).
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Figure 1: IP6 is required for NHEJ in vitro. (A) Structure of IP6. With d-myo-inositol as its base ring structure, IP6 has one axial phosphate group (C-2), five phosphate groups in equatorial position and a molecular weight of 660 Da. (B) End-joining assays were carried out using 20 µg of HeLa WCE or AS65 as described in Materials and Methods section and complemented with IP6 or IS6. Treatment with Wortmannin (3 μM, lanes 4 and 10) and anti-XRCC4 antibodies (α-XRCC4, 1:250 dilution, lanes 3 and 9) demonstrate that the observed end joining is bona fide NHEJ. A −ve sign indicates no protein. Monomer, monomeric DNA substrate; dimer, trimer, etc., end-joined concatamers. (C) IP6 increased the rate of NHEJ in vitro. AS65 (20 µg) was assayed for end joining as described in Materials and Methods section in the presence (left) or absence (right) of 1 μM IP6 for the indicated time. (D) Quantification of C presented as percent of ends joined as a function of reaction time. Graph includes controls in which reactions were treated with anti-XRCC4 antibodies (1:250 dilution) to inhibit NHEJ (filled triangle, + IP6 + α-XRCC4; open triangle, − IP6 + α-XRCC4), which are not shown in (C).
Mentions: We have shown that, in addition to activating DNA-PKcs, Ku binds the small, phosphate-rich molecule inositol hexakisphosphate (IP6, Figure 1A), which we previously identified as a stimulatory factor in mammalian NHEJ (9,10). Subsequent publications demonstrated that binding of IP6 by Ku did not change the ability of Ku to bind DNA or to assemble with DNA-PKcs to form DNA-PK (10,11). These data suggest that the role of IP6 in mammalian NHEJ is tied to functions of Ku that are independent of DNA-PK. To determine if binding of IP6 by Ku contributes to efficient NHEJ, we used mutational analysis to identify an IP6-binding site in Ku. This mutational analysis revealed that the Ku IP6-binding site, like its DNA-binding site, is bipartite and requires residues from both Ku70 and Ku80 subunits. IP6-binding mutants of Ku were separation-of-function mutants that stimulated DNA-PK but showed reduced affinity for IP6. These separation-of-function mutants were also impaired for complementation of NHEJ in Ku-depleted extracts, which demonstrates that formation of a Ku–IP6 complex is important for efficient NHEJ in vitro. These data show that the role of Ku in mammalian NHEJ may be divided into DNA-PKcs-dependent and IP6-dependent functions.Figure 1.

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