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.2014 Mar 3;33(5):482-500.
doi: 10.1002/embj.201386100. Epub 2014 Feb 3.

ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling

Affiliations

ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling

Rajashree A Deshpande et al. EMBO J..

Erratum in

Abstract

The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

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Figures

Figure 1
Figure 1
ATP induces conformational changes in Mre11 and Rad50.

  1. Schematic of Mre11 (blue) and Rad50 (orange, yellow) catalytic domains and conformational changes upon ATP binding.

  2. MR solution structure. Experimental SAXS data of ATP-boundP. furiosus Mre11-Rad50cd-linked in solution closely matches calculated SAXS scattering curves of MRcd crystal structures in nucleotide-bound states (example shown inset).

  3. Rad50 colored as follows: N-terminal lobe, blue; signature coupling helices, cyan; C-terminal lobe, orange; extended signature motif, magenta; core cavity, green surface. The cavity is remodeled as Rad50 rotates 35° between nucleotide states. See also Supplementary Movie 1.

  4. Close-up of Rad50 core cavity, basic-switch and core Leu residue changes between nucleotide-free (left) and nucleotide-bound (right) states. Colored as in (C), also see Supplementary Movie 2.

Figure 2
Figure 2
Rad50 cavity alterations destabilize the ATP-bound state.

  1. Gel filtration of Rad50cd-linked proteins with or without ATP. Lines across peaks represent molecular weights measured by MALS (right axis); arrows show positions of dimer peaks. See also Supplementary Fig S1.

  2. SAXS time-course assays showing scattering curves of MRcd WT, L802W, or R805E variants with 0.5 mM ATP at 65°C, plotted at the indicated time points.

  3. SAXS similarity matrices were made by comparing MRcd variant scattering curves for each time point in the SAXS time-course experiment, measured with ATP at 65°C, compared to the 0 min curve. As indicated by the scale, blue represents very high and red represents very low similarity of curves based on χ2 scores.

  4. SAXS analysis of WT and L802W MRcd complexes in the presence of 0.5 mM ATP or ATPγS at 65°C as indicated.

  5. The L802W Rad50cd-linked structure, colored as in Fig 1C, is superimposed on WT Rad50cd-linked, with transparent coloring and cavity (dotted surface). The Trp substitution overlaps with the WT surface, remodeling the Rad50 core.

  6. ATP-driven conformational states. Superimposition of nucleotide-free Rad50 (blue) with nucleotide-bound Rad50 (gray, left) and nucleotide-free L802W Rad50 (orange, right) using C-terminal domain residues 741–785 for the alignment. N-terminal lobes are shown for clarity.

Figure 3
Figure 3
ATP binding promotes conformational changes in Rad50 that are measurable by partial proteolysis. A  Rad50cd (5 μg) proteolysis with indicated nucleotides revealing distinct conformational states. Triangles show ATP-stimulated products of proteolysis at K155 (yellow triangle) and K171 (black triangle). B  Proteolysis of Rad50cd-linked (5 μg) variants with ATP as in (A). C  Proteolysis of Rad50cd-linked L802W (5 μg) with indicated nucleotides as in (A). D, E  Proteolysis of full-length WT or mutant MR (1 μg Rad50) with nucleotide. Red triangle indicates K63 cleavage product. Quantitated levels of undigested protein are shown below each lane. Also see Supplementary Fig S3 and Supplementary Table S1. F  Structural models of the ATP-free and ATP-bound MRcd generated from Mre11-Rad50 structures from PDB ID 3QKS (open) and PDB ID 3QKU (closed) superimposed withT. maritima MR (PDB ID 3QG5; open) andM. jannaschii MR (PDB ID 3AV0; closed). Locations of tryptic digest sites are mapped, with two views of the closed model shown for clarity. Residues 327 to 357 of Mre11 are in the linker region; residues 394–404 are outside of the region that was crystallized. Source data are available online for this figure.
Figure 4
Figure 4
Rad50 catalytic domain mutations affect ATP-dependent DNA binding and hydrolysis.

  1. ATP binding by full-length MR complexes as measured by quenching of tryptophan fluorescence by TNP-ATP. Shown are emission scans using 290 nm excitation and TNP-ATP as indicated.

  2. Quantitation of quenching efficiency (F0/F) at 330 nm emission calculated from (A).

  3. ATPase kinetics measurements of Rad50cd-linked and full-length MR proteins (see also Supplementary Fig S4).

  4. Gel-shift assays of Rad50cd-linked (2.5 μM) with a 41-bp DNA duplex substrate.

  5. Gel-shift assays of MRcd complexes (400 nM) with a 41-bp DNA duplex substrate. Protein-DNA complexes are indicated (I and II), with the upper band (I) being nucleotide-dependent.

  6. Gel-shift assays as in (E) with full-length MR (200 nM).

  7. Crystal structures reveal hydrogen-bonding rearrangements between nucleotide-free WT (left) and R805E Rad50cd-linked (right). Colored as in Fig C, with the K54 loop in burgundy and red arrows highlighting major changes. See also Supplementary Fig S4.

Source data are available online for this figure.
Figure 5
Figure 5
DNA end tethering by MR and designed mutants.

  1. Full-length MR (50 nM) was assayed for DNA end tethering with pCDF-1b plasmid, nucleotide, andE. coli DNA ligase (NAD-dependent) as indicated.

  2. Assays were performed as in (A) with Rad50 (50 nM or as indicated) and Mre11.

  3. Assays were performed as in (A) with Rad50cd-linked.

  4. Assays were performed as in (A) with MRcd.

  5. Assays were performed as in (A) with full-length wt MR and nuclease-deficient MR (Mre11 H85L).

  6. Assays were performed as in (A) with MRcd Rad50 variants as indicated.

  7. Assays were performed as in (A) with full-length MR Rad50 variants.

  8. DNA end tethering assays were performed as in (A) with the indicated addition of supercoiled pCDF-1b competitor DNA.

Data information: Reactions in lanes 4, 6, 10, and 14: substrate added before competitor; reactions in lanes 7, 8, 11, 12, 15, and 16: competitor added before substrate (* indicates positions of competitor DNA). Numbers indicate relative amount of competitor vs. substrate DNA. Ligation products (circular and multimer-linear) are indicated. Source data are available online for this figure.
Figure 6
Figure 6
Rad50 separation-of-function mutations affect MR nuclease activity.

  1. Endonuclease assay for full-length MR variants (300 and 600 nM) on a 717-bp double-stranded DNA substrate. The shorter oligonucleotides are endonucleolytic products, except bands marked with *.

  2. Nuclease assay as in (A), but with a 41-bp double-stranded DNA substrate.

  3. Nuclease assay as in (A), with WT full-length MR (0.9 μM) and MRcd (1 and 2.5 μM).

  4. Exonuclease assay for full-length MR variants (100 and 200 nM) using a 41-bp double-stranded DNA, labeled on the 5′ end of the top strand with32P (*).

  5. Resection assays with full-length MR (nM concentrations as indicated), HerA and NurA (HN) with a 3.8-kb linearized plasmid.

Source data are available online for this figure.
Figure 7
Figure 7
Cross-linking of Rad50 ATPase domains blocks DNA end resection.

  1. Crosslinking scheme uses introduced cysteines that are brought close enough to form disulfide bonds when Rad50 dimerizes with ATP.

  2. Crosslinking of Rad50cd-2C variants (3.5 μM) with indicated nucleotides (0.5 mM ATP or 2 mM other nucleotides), separated by SDS–PAGE in the absence of reducing agent except in lanes marked ‘βME’.

  3. Full-length MR or MR-2C proteins (2.35 μM) were cross-linked with H2O2 (2 mM) with DTT (mM) and ATP (0.5 mM) as indicated.

  4. Cross-linked full-length MR and MR-2C (nM) were assayed for DNA end tethering as in Fig 5A with H2O2 (2 mM), DTT (mM) and T4 DNA ligase added as shown.

  5. MR-2C (1.6 μM) and full-length MR (0.9 μM) were tested for endonuclease activity as in Fig 6A following crosslinking with DTT (mM) and H2O2 (2 mM) as indicated.

  6. MR-2C and full-length MR (nM) were cross-linked with DTT (mM) and H2O2 (2 mM) as indicated and assayed for plasmid resection as in Fig 6D. Also see Supplementary Fig S5.

Source data are available online for this figure.
Figure 8
Figure 8
Rad50 separation-of-function mutations affect MRN DNA repair functions. A  S. cerevisiae survival assays withrad50 S. cerevisiae strains expressing vector only or WT, I1214W, or R1217E Rad50 proteins. B  Diagram ofS. cerevisiae SSA assay (left)(Vazeet al, 2002). Survival ofrad50 S. cerevisiae strains expressing vector only or WT, I1214W, or R1217E Rad50 proteins before (glucose) or after (galactose) DSB induction atleu2::cs. C  Chromosomal NHEJ assay results withS. cerevisiae Rad50 variant strains, characterizing precise versus imprecise NHEJ-mediated joining events. See Supplementary Fig S6 for assay details. The average of 3 independent experiments is shown, with error bars indicating standard deviation. D  NHEJ assay with EcoRI induction, causing genome-wide DSBs. WT, I1214W, or R1217E alleles of Rad50 were expressed in arad50 strain and survival was measured after galactose induction of EcoRI for 8 hrs. The average of 5 independent experiments is shown, with the survival of each strain shown relative to therad50 strain that was normalized to 1; error bars indicate standard deviation. E  S. pombe survival assays of Rad50 variant strains. F, G  Western blots for γH2A and H2A levels in cell lysates after 90 Gy IR treatment of indicatedS. pombe strains. H  Stimulation of ATM phosphorylation of p53 by the human MRN complex in vitro. Blots were probed with antibody against phospho-Ser15 of p53. I  Model of ATP-induced conformational changes in MR and the functions associated with each state. See text for details. Source data are available online for this figure.
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References

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