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Phosphorylation in the serine/threonine 2609-2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts.

Povirk LF, Zhou RZ, Ramsden DA, Lees-Miller SP, Valerie K - Nucleic Acids Res. (2007)

Bottom Line: The residual end joining was greater with the S/T --> D-substituted than with the S/T --> A-substituted protein.The fidelity of end joining was not affected by the mutant DNA-PKcs alleles or the inhibitors.Overall, the results support a role for autophosphorylation of the 2609-2647 cluster in promoting end joining and controlling the accessibility of DNA ends, but suggest that DNA-PK-mediated phosphorylation at other sites, on either DNA-PKcs or other proteins, is at least as important as the 2609-2647 cluster in regulating end joining.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA. LPOVIRK@vcu.edu

ABSTRACT
Previous work suggested that phosphorylation of DNA-PKcs at several serine/threonine (S/T) residues at positions 2609-2647 promotes DNA-PK-dependent end joining. In an attempt to clarify the role of such phosphorylation, end joining was examined in extracts of DNA-PKcs-deficient M059J cells. Joining of ends requiring gap filling prior to ligation was completely dependent on complementation of these extracts with exogenous DNA-PKcs. DNA-PKcs with either S/T --> A or S/T --> D substitutions at all six sites in the 2609-2647 cluster also supported end joining, but with markedly lower efficiency than wild-type protein. The residual end joining was greater with the S/T --> D-substituted than with the S/T --> A-substituted protein. A specific inhibitor of the kinase activity of DNA-PK, KU57788, completely blocked end joining promoted by wild type as well as both mutant forms of DNA-PK, while inhibition of ATM kinase did not. The fidelity of end joining was not affected by the mutant DNA-PKcs alleles or the inhibitors. Overall, the results support a role for autophosphorylation of the 2609-2647 cluster in promoting end joining and controlling the accessibility of DNA ends, but suggest that DNA-PK-mediated phosphorylation at other sites, on either DNA-PKcs or other proteins, is at least as important as the 2609-2647 cluster in regulating end joining.

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Promotion of end joining of a substrate with partially complementary 3′ overhangs by wild-type and mutant alleles of DNA-PKcs. The substrate shown in Figure 1 was incubated for 6 h in whole-cell extracts supplemented with wild-type DNA-PKcs (wt), or DNA-PKcs with 6 S/T → A (A6) or S/T → D (D6) substitutions in the ABCDE cluster. Products were then analyzed on denaturing sequencing gels. See Figure 1 for definition of substrates and products. (A) End joining in presence of 2, 4 or 8 nM of each DNA-PKcs protein. (B) Total accurate end joining (42-mer plus 24-mer product) was calculated for the wt (filled circle), A6(filled square) and D6 (filled triangle) alleles at each concentration of DNA-PKcs. Error bars represent range of values in two independent experiments. (C) Effect of KU57788 (1 μM) on end joining and gap filling by each DNA-PKcs allele (4 nM). Some samples contained 100 μM ddTTP in place of dTTP, as indicated. The labeled single-stranded 15-mer fragment (released from the top strand of the plasmid by TaqI) represents addition of an unligatable ddTMP nucleotide to the -AGC overhang in the labeled top strand. (D) Phosphorimage intensity profiles from the gel shown in (C), in the area of the 42-base end-joining product, for the lanes containing samples with the A6 and D6 mutant alleles, without ddTTP. The profiles for the samples with and without KU57788 have been superimposed. In both cases, there is clearly a peak of 42-base product that is completely eliminated by KU57788.
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Figure 3: Promotion of end joining of a substrate with partially complementary 3′ overhangs by wild-type and mutant alleles of DNA-PKcs. The substrate shown in Figure 1 was incubated for 6 h in whole-cell extracts supplemented with wild-type DNA-PKcs (wt), or DNA-PKcs with 6 S/T → A (A6) or S/T → D (D6) substitutions in the ABCDE cluster. Products were then analyzed on denaturing sequencing gels. See Figure 1 for definition of substrates and products. (A) End joining in presence of 2, 4 or 8 nM of each DNA-PKcs protein. (B) Total accurate end joining (42-mer plus 24-mer product) was calculated for the wt (filled circle), A6(filled square) and D6 (filled triangle) alleles at each concentration of DNA-PKcs. Error bars represent range of values in two independent experiments. (C) Effect of KU57788 (1 μM) on end joining and gap filling by each DNA-PKcs allele (4 nM). Some samples contained 100 μM ddTTP in place of dTTP, as indicated. The labeled single-stranded 15-mer fragment (released from the top strand of the plasmid by TaqI) represents addition of an unligatable ddTMP nucleotide to the -AGC overhang in the labeled top strand. (D) Phosphorimage intensity profiles from the gel shown in (C), in the area of the 42-base end-joining product, for the lanes containing samples with the A6 and D6 mutant alleles, without ddTTP. The profiles for the samples with and without KU57788 have been superimposed. In both cases, there is clearly a peak of 42-base product that is completely eliminated by KU57788.

Mentions: Figure 3 shows end joining of the 1-base-gapped substrate in extracts supplemented with these wild-type and mutant DNA-PKcs proteins. DNA-PKcs-D6, designed to mimic constitutive phosphorylation of the ABCDE cluster, supported end joining, but with markedly lower efficiency than the wild-type protein (Figure 3A and B). The S/T → D mutations did not affect the fidelity of end joining, as the accurate 42- and 24-base products were formed almost exclusively. DNA-PKcs-A6 also supported end joining, but even less efficiently than the D6 mutant. Nevertheless, even at the lowest concentration of DNA-PKcs-A6, joining was significantly greater than that seen with no DNA-PKcs (0.5% versus <0.05%). Moreover, in three replicate experiments, the difference between the D6 and A6 alleles was consistently more pronounced at 8 nM than at 2–4 nM (Figure 3B).Figure 3.


Phosphorylation in the serine/threonine 2609-2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts.

Povirk LF, Zhou RZ, Ramsden DA, Lees-Miller SP, Valerie K - Nucleic Acids Res. (2007)

Promotion of end joining of a substrate with partially complementary 3′ overhangs by wild-type and mutant alleles of DNA-PKcs. The substrate shown in Figure 1 was incubated for 6 h in whole-cell extracts supplemented with wild-type DNA-PKcs (wt), or DNA-PKcs with 6 S/T → A (A6) or S/T → D (D6) substitutions in the ABCDE cluster. Products were then analyzed on denaturing sequencing gels. See Figure 1 for definition of substrates and products. (A) End joining in presence of 2, 4 or 8 nM of each DNA-PKcs protein. (B) Total accurate end joining (42-mer plus 24-mer product) was calculated for the wt (filled circle), A6(filled square) and D6 (filled triangle) alleles at each concentration of DNA-PKcs. Error bars represent range of values in two independent experiments. (C) Effect of KU57788 (1 μM) on end joining and gap filling by each DNA-PKcs allele (4 nM). Some samples contained 100 μM ddTTP in place of dTTP, as indicated. The labeled single-stranded 15-mer fragment (released from the top strand of the plasmid by TaqI) represents addition of an unligatable ddTMP nucleotide to the -AGC overhang in the labeled top strand. (D) Phosphorimage intensity profiles from the gel shown in (C), in the area of the 42-base end-joining product, for the lanes containing samples with the A6 and D6 mutant alleles, without ddTTP. The profiles for the samples with and without KU57788 have been superimposed. In both cases, there is clearly a peak of 42-base product that is completely eliminated by KU57788.
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Related In: Results  -  Collection

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Figure 3: Promotion of end joining of a substrate with partially complementary 3′ overhangs by wild-type and mutant alleles of DNA-PKcs. The substrate shown in Figure 1 was incubated for 6 h in whole-cell extracts supplemented with wild-type DNA-PKcs (wt), or DNA-PKcs with 6 S/T → A (A6) or S/T → D (D6) substitutions in the ABCDE cluster. Products were then analyzed on denaturing sequencing gels. See Figure 1 for definition of substrates and products. (A) End joining in presence of 2, 4 or 8 nM of each DNA-PKcs protein. (B) Total accurate end joining (42-mer plus 24-mer product) was calculated for the wt (filled circle), A6(filled square) and D6 (filled triangle) alleles at each concentration of DNA-PKcs. Error bars represent range of values in two independent experiments. (C) Effect of KU57788 (1 μM) on end joining and gap filling by each DNA-PKcs allele (4 nM). Some samples contained 100 μM ddTTP in place of dTTP, as indicated. The labeled single-stranded 15-mer fragment (released from the top strand of the plasmid by TaqI) represents addition of an unligatable ddTMP nucleotide to the -AGC overhang in the labeled top strand. (D) Phosphorimage intensity profiles from the gel shown in (C), in the area of the 42-base end-joining product, for the lanes containing samples with the A6 and D6 mutant alleles, without ddTTP. The profiles for the samples with and without KU57788 have been superimposed. In both cases, there is clearly a peak of 42-base product that is completely eliminated by KU57788.
Mentions: Figure 3 shows end joining of the 1-base-gapped substrate in extracts supplemented with these wild-type and mutant DNA-PKcs proteins. DNA-PKcs-D6, designed to mimic constitutive phosphorylation of the ABCDE cluster, supported end joining, but with markedly lower efficiency than the wild-type protein (Figure 3A and B). The S/T → D mutations did not affect the fidelity of end joining, as the accurate 42- and 24-base products were formed almost exclusively. DNA-PKcs-A6 also supported end joining, but even less efficiently than the D6 mutant. Nevertheless, even at the lowest concentration of DNA-PKcs-A6, joining was significantly greater than that seen with no DNA-PKcs (0.5% versus <0.05%). Moreover, in three replicate experiments, the difference between the D6 and A6 alleles was consistently more pronounced at 8 nM than at 2–4 nM (Figure 3B).Figure 3.

Bottom Line: The residual end joining was greater with the S/T --> D-substituted than with the S/T --> A-substituted protein.The fidelity of end joining was not affected by the mutant DNA-PKcs alleles or the inhibitors.Overall, the results support a role for autophosphorylation of the 2609-2647 cluster in promoting end joining and controlling the accessibility of DNA ends, but suggest that DNA-PK-mediated phosphorylation at other sites, on either DNA-PKcs or other proteins, is at least as important as the 2609-2647 cluster in regulating end joining.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA. LPOVIRK@vcu.edu

ABSTRACT
Previous work suggested that phosphorylation of DNA-PKcs at several serine/threonine (S/T) residues at positions 2609-2647 promotes DNA-PK-dependent end joining. In an attempt to clarify the role of such phosphorylation, end joining was examined in extracts of DNA-PKcs-deficient M059J cells. Joining of ends requiring gap filling prior to ligation was completely dependent on complementation of these extracts with exogenous DNA-PKcs. DNA-PKcs with either S/T --> A or S/T --> D substitutions at all six sites in the 2609-2647 cluster also supported end joining, but with markedly lower efficiency than wild-type protein. The residual end joining was greater with the S/T --> D-substituted than with the S/T --> A-substituted protein. A specific inhibitor of the kinase activity of DNA-PK, KU57788, completely blocked end joining promoted by wild type as well as both mutant forms of DNA-PK, while inhibition of ATM kinase did not. The fidelity of end joining was not affected by the mutant DNA-PKcs alleles or the inhibitors. Overall, the results support a role for autophosphorylation of the 2609-2647 cluster in promoting end joining and controlling the accessibility of DNA ends, but suggest that DNA-PK-mediated phosphorylation at other sites, on either DNA-PKcs or other proteins, is at least as important as the 2609-2647 cluster in regulating end joining.

Show MeSH
Related in: MedlinePlus