Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (2), eaay0922
eCollection

DNA-dependent Protein Kinase Promotes DNA End Processing by MRN and CtIP

Affiliations

DNA-dependent Protein Kinase Promotes DNA End Processing by MRN and CtIP

Rajashree A Deshpande et al. Sci Adv.

Abstract

The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells-a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.

Figures

Fig. 1
Fig. 1. Nucleolytic removal of DNA-PK by MRN and its stimulation by CtIP.
(A) Nuclease reactions were performed with a 197-bp DNA substrate, 5′ labeled with 32P (asterisk), with MRN (50 nM), CtIP (80 nM), Ku (10 nM), and DNA-PKcs (10 nM) as indicated, in the presence of both magnesium and manganese (lanes 1 to 8) or magnesium only (lanes 9 to 16). All reactions contained the DNA-PKcs inhibitor NU7441. Products were visualized by denaturing PAGE and visualized by phosphorimager. Red and black arrows indicate the predominant product in the presence of DNA-PKcs and Ku, or Ku alone, respectively. (B) Nuclease assays were performed as in (A) in the presence of both magnesium and manganese with wild-type MRN or MRN containing nuclease-deficient Mre11 (H129N). Reactions without ATP or with AMP-PNP instead of ATP are indicated, as is the reaction without NU7441. (C) Nuclease assays were performed as in (A) in the presence of magnesium, manganese, ATP, NU7441, CtIP (C), and DNA-PK (D) with MRN (M) containing wild-type Rad50 (WT) or adenosine triphosphatase (ATPase)–deficient Rad50 K42A (KA) or D1231A (DA). (D) Nuclease assays were performed as in (A) in the presence of both magnesium and manganese with wild-type MRN in the presence of 25, 50, and 100 μM Mre11 inhibitors and NU7441. (E) Quantitation of the MRN endonuclease observed in the presence of Mre11 inhibitors expressed as percentage of the activity in the absence of inhibitors. Error bars represent SD from two replicates.
Fig. 2
Fig. 2. MRN, DNA-PK, and CtIP promote dsDNA end resection.
(A) A linear map of CtIP indicating a subset of known phosphorylated residues as well as residues important for DNA binding and catalytic activity as discussed in the text (orange, sites required for nuclease activity; green, ATM-dependent phosphorylation sites; blue, CDK-dependent phosphorylation sites; red, ATR/ATM-dependent phosphorylation site). (B) Nuclease assays were performed with MRN (12.5 nM), CtIP (40 nM), Ku (10 nM), DNA-PKcs (10 nM), and NU7441 as in Fig. 1A but with various mutants of CtIP, as indicated, in the presence of both magnesium and manganese. The red arrow indicates the predominant product formed in the presence of DNA-PKcs. (C) Nuclease assays were performed as in (B) with titrations of CtIP (10, 20, and 40 nM). (D) DNA end resection on a plasmid substrate (3.6 kb) was performed with MRN, CtIP, DNA-PK, and Exo1, as indicated, in the presence of a DNA-PKcs inhibitor. Reaction products were separated in a native agarose gel, which was stained with SYBR Green; molecular weight (MW) ladder migration is shown (kb). (E) dsDNA cleavage products from the MRN nuclease assay with DNA-PK and CtIP were detected on a 12% native polyacrylamide gel. The red arrow indicates the ~45-bp product. (F) Protein-protein interactions between CtIP, MRN, and DNA-PK were measured by IP with anti-CtIP antibody in the presence or absence of ATP followed by Western blotting of bound proteins, as indicated. (G) Interactions between CtIP, MRN, DNA-PKcs, and Ku were measured with CtIP IP as in (F) in the presence of ATP.
Fig. 3
Fig. 3. Single-molecule visualization of DNA-PK on DNA.
(A) Schematic of the DNA curtains assay for DNA-PK. (B) Illustration and kymograph (time series of one molecule over the course of the reaction) of DNA-PKcs injection onto DNA curtains. White arrow indicates a single DNA-PKcs binding event. This molecule then slides along the DNA in the direction of buffer flow to reach the DNA end (green arrow). The molecule then associates for a short time at the end (te) before dissociation (red arrow). (C) Lifetime of DNA-PKcs on DNA curtains in the presence of ATP. (D) Illustration and kymographs of DNA-PKcs colocalizing with an end-bound Ku and Ku(5A) in the presence or absence of ATP as indicated. (E and F) Lifetime of Ku (WT or 5A) on DNA curtains in the presence or absence of DNA-PKcs or ATP as indicated. Table shows half-life of Ku(WT) or Ku(5A) under various conditions and the number of molecules observed (N).
Fig. 4
Fig. 4. Single-molecule visualization of DNA-PK removal by MRN and CtIP.
(A) Illustration and kymographs of DNA-PKcs (magenta) and Ku (green) upon injection of MRN and CtIP [both unlabeled (dark)]. (B) Kymographs of DNA-PKcs (magenta) with Ku (unlabeled), with injection of MRN (green) alone (top), or MRN with CtIP (middle), or a nuclease-dead mutant of MRN (H129N) with CtIP (bottom). (C and D) Lifetimes and associated half-lives of the DNA-PK complex (as observed by DNA-PKcs occupancy) upon no injection (green), injection with MRN and CtIP (black), MRN alone (red), nuclease-deficient H129N MRN and CtIP (magenta), CtIP only (blue), or MRN with phospho-blocking T847A/T859A CtIP (purple). Table shows half-life of DNA-PKcs under various conditions and the number of molecules observed (N). (E) Schematic model for DNA-PK removal from DSB ends, as discussed in the text. DNA-PKcs binds to the Ku heterodimer bound to DNA at the DSB. Transition from NHEJ to homologous recombination (HR) results from (i) DNA-PKcs phosphorylation of Ku, resulting in dissociation of Ku as well as DNA-PKcs from ends, (ii) DNA-bound DNA-PK stimulation of MRN single-strand endonucleolytic cleavage followed by 5′ to 3′ resection, or (iii) DNA-PK stimulation of MRN double-stranded endonucleolytic cleavage resulting in DNA-PK loss and 5′ to 3′ resection. Long-range 5′ to 3′ resection ensues from the nick or a new DSB, creating 3′ single-stranded DNA that is used for homology search. The DNA-bound DNA-PK complex (dashed box) is the species isolated in the modified ChIP protocol (Fig. 5 and fig. S4).
Fig. 5
Fig. 5. DNA-PK–associated DNA end fragments are observed at AsiSI breaks in human cells using GLASS-ChIP.
(A) Small dsDNA products resulting from nucleolytic cleavage of DNA-PK–bound AsiSI–generated DNA ends (dashed line box, Fig. 4E) were isolated from U2OS cells treated with 4-OHT or vehicle for 4 hours as indicated. NU7441 (10 μM) was added as indicated for 5 hours starting at 1 hour before 4-OHT addition to induce AsiSI. DNA-PK–bound DNA was isolated using a modified ChIP protocol (GLASS-ChIP, fig. S4) and quantified by qPCR using primers located ~30 nt from the AsiSI cut site (results from primers ~300 nt from cut sites in fig. S5). Primer set U1 (solid) is upstream, whereas D1 (checkered) is downstream of the AsiSI cut sites. The DNA quantitated from U2OS cells in the presence or absence of 4-OHT and a DNA-PKcs inhibitor (NU7441) is shown for each AsiSI site (46). The inset magnifies the results for experiments performed in the absence of NU7441. Results are from three independent biological replicates, with Student’s two-tailed t test performed; *P < 0.05, ** P < 0.01, ***P < 0.001, in comparison to equivalent samples without 4-OHT. (B) The GLASS-ChIP protocol was performed as in (A) using cells treated with a DNA-PKcs inhibitor (NU7441, 10 μM), a Mre11 inhibitor (PFM03, 100 μM), and 4-OHT for 1 hour as indicated. Results are from three independent biological replicates, with Student’s two-tailed t test performed; **P < 0.005 and **** P < 0.0001, in comparison to equivalent samples without PFM03.

Similar articles

See all similar articles

References

    1. Jette N., Lees-Miller S. P., The DNA-dependent protein kinase: A multifunctional protein kinase with roles in DNA double strand break repair and mitosis. Prog. Biophys. Mol. Biol. 117, 194–205 (2015). - PMC - PubMed
    1. Chang H. H. Y., Pannunzio N. R., Adachi N., Lieber M. R., Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat. Rev. Mol. Cell Biol. 18, 495–506 (2017). - PubMed
    1. Goodarzi A. A., Jeggo P. A., The repair and signaling responses to DNA double-strand breaks. Adv. Genet. 82, 1–45 (2013). - PubMed
    1. Symington L. S., Mechanism and regulation of DNA end resection in eukaryotes. Crit. Rev. Biochem. Mol. Biol. 51, 195–212 (2016). - PMC - PubMed
    1. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W., Jeggo P. A., Jackson S. P., Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80, 813–823 (1995). - PubMed

Publication types

Feedback