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

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.

Figure 1.

Requirement for catalytically active DNA-PK in end joining of a substrate with partially complementary 3′ overhangs. The internally labeled (asterisk) substrate shown (10 ng) was incubated for 6 h in whole-cell extracts (120 μg) or nuclear extracts (30 μg) from DNA-PKcs-deficient M059J cells. Extracts were supplemented with purified human DNA-PKcs from HeLa cells and/or recombinant X4L4 as indicated. DNA was then cut with BstXI and TaqI and analyzed on denaturing sequencing gels. (A) Internally labeled substrate and expected end-joining products. The bolded ‘T’ indicates fill-in of the 1 base gap in each strand of the aligned ends. (B) End joining by whole-cell extracts supplemented with 0, 3, 6 or 10 nM DNA-PKcs, or nuclear extracts supplemented with 0, 3 or 6 nM DNA-PKcs plus 20 nM X4L4 as indicated. (C) Inhibition of end joining in the presence of 6 nM DNA-PKcs by the DNA-PK inhibitors KU57788 (1 μM) and wortmannin (3 μM). Samples contained 1.2% DMSO as indicated. (D) Effect of the ATM inhibitor KU55933; same conditions as (C) except that all samples contained 1.2% DMSO. (E) Quantitative data from experiments similar to that shown in (D); the extent of accurate end joining was calculated as the sum of the 24- and 42-base fragments divided by the total phosphorimage intensity in each lane, then normalized to the end joining seen in the presence of DNA-PKcs and DMSO without any inhibitor. Error bars indicate SEM of four measurements, each from a separate experiment except the 10 μM point, which was from two measurements.

Figure 2.

Requirement for catalytically active DNA-PK in end joining of a substrate with cohesive 5′ overhangs. MluI-cut pRZ56 was 5′-³²P-end-labeled (asterisk) and incubated for 1 h in whole-cell extracts containing 6 nM purified DNA-PKcs and inhibitors as indicated. (A) Denaturing sequencing gel showing products formed. (B) Agarose gel showing titration of inhibition by KU57788. (C) Quantitation of inhibition of end joining of the cohesive end substrate by KU57788 (filled circle) or by the ATM inhibitor KU55933 (filled square). Error bars show range of values obtained in two independent experiments. A single titration for KU57788 with the partially complementary substrate (see Figure 1) is shown for comparison (open triangle). Note that since a single joining event results in both the joined and unjoined ends of a single plasmid migrating as a dimer, the agarose gel assay results in higher apparent levels of end joining than the sequencing gel assay.

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.

Figure 4.

Promotion of end joining of a cohesive-end substrate by wild-type and mutant alleles of DNA-PKcs. The substrate shown in Figure 2 was endlabeled and incubated for 1 h in extracts supplemented with the wt, A6 and D6 DNA-PKcs alleles. (A) Agarose gel analysis of end joining in the presence of each DNA-PKcs allele at 0, 2, 4 or 8 nM. (B) Concentration dependence of end joining with the wild-type (filled circle), A6(filled square) and D6 (filled triangle) alleles. Error bars show range of values obtained in two independent experiments when larger than the symbols; the 13 nM point was from a single experiment due to the large amount of DNA-PKcs required. (C) Effect of KU57788 (1 μM) on end joining with each DNA-PKcs allele (4 nM). (D) Same as C., except analysis was by BstXI/TaqI cleavage and denaturing sequencing gel electrophoresis.