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Eukaryotic Rad50 functions as a rod-shaped dimer

Nature Structural & Molecular Biology volume 24, pages 248–257 (2017)Cite this article

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Abstract

The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 molecules interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we determined the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.

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Figure 1: Rod-shaped structure of the Zn2+-bound human Rad50 apex dimer.
Figure 2: Two interfaces at the apex of the HsRad50 dimer stabilize the rod-shaped structure.
Figure 3: Solution structure of the HsRad50 apex dimer.
Figure 4: FRET and cross-linking analysis of wild-type and mutant Rad50HCC.
Figure 5: DSB repair and Mre11-complex integrity of Rad50 coiled-coil and hook-dimerization interface mutants.
Figure 6: Effect of Rad50 coiled-coil and hook interface mutants on the Mre11 complex's role in Tel1/ATM checkpoint signaling, meiosis and associated biochemical activities.
Figure 7: The Rad50 rod-shaped coiled-coil structure at the apex is important for Mre11-complex globular domain functions.

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Acknowledgements

We thank T. Kochańczyk for discussions and information regarding fluorescent protein modification, and members of the Cho and Petrini labs for helpful comments throughout. Expression constructs for wild-type MRN were gifts from T. Paull (University of Texas, Austin, Texas, USA). This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korea Government (MEST, grant 2015R1A2A1A05001694 to Y.C.), the BK21 Program (Ministry of Education) (to Y.C.), the NIH (RO1 grant GM56888 to J.H.J.P.), the MSK Cancer Center (Core Grant P30 CA008748 to J.H.J.P.), and the National Science Center, Poland (Opus, grant 2014/13/B/NZ1/ 00935 to A.K.).

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  1. Young Bong Park and Marcel Hohl: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Life Sciences, Pohang University of Science and Technology, Pohang, South Korea

    Young Bong Park, Eunyoung Jeong & Yunje Cho

  2. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Marcel Hohl & John H J Petrini

  3. Laboratory of Chemical Biology, University of Wrocław, Wrocław, Poland

    Michał Padjasek & Artur Krężel

  4. Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, South Korea

    Kyeong Sik Jin

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Contributions

Y.B.P. carried out crystallization and structure determination; Y.B.P., M.P., M.H. and E.J. participated in biochemical experiments; K.S.J. performed SAXS analyses; M.H. and J.H.J.P. designed and performed yeast genetics experiments; Y.C. and J.H.J.P. designed research in consultation with A.K.; and J.H.J.P., Y.C., A.K. and M.H. wrote the manuscript.

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Correspondence to John H J Petrini or Yunje Cho.

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Integrated supplementary information

Supplementary Figure 1 Structure of the human and yeast Rad50 hook and coiled-coil domains.

(a) Analytical ultracentrifuge data of the Zn-free (bottom left) and –bound Rad50HCC182 constructs (bottom right). (b) Stereoview of the section of the electron density map of Rad50HCC182. The 2.8 Å experimental map contoured at 1.2 I/σ was generated with phases from derived Se-Met anomalous scattering. (c) Sequence alignment of Rad50 homologs. Secondary structures of the coiled-coil domain of HsRad50 are represented above the sequence. Secondary structures of PfRad50 are shown below the sequence of PfRad50. Conserved residues in Rad50 proteins are marked with a yellow box. Mutations identified in breast cancer patients are marked with a blue star. Residues involved in dimerization are marked with a violet square. Rad50 hook mutant suppressors20 are marked with a green triangle. Every tenth residue is marked with a black circle. (d-e) A model for ScRad50 apex dimer. (d) The model was generated using the program Modeller (Sali, A. & Blundell, T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993)) based on human Rad50 apex structure. Close up view of the hook interface of ScRad50 shown in the same orientation as in Fig. 2a. (e) Close up view of the hook and coiled-coil interfaces of ScRad50. The view is orthogonal from that of Supplementary Fig.1d. (f) Close up view of the coiled-coil interface of ScRad50.

Supplementary Figure 2 The Rad50 ‘hinge’ loop and hook domain cooperate to promote DSB repair and Mre11-complex integrity.

(a) Close up view of the hinge loop in Rad50 A (top, blue) and Rad50B (bottom, orange). The three mutated residues are highlighted in yellow. (b, c) DNA damage sensitivity of the mutants towards MMS (b) and CPT (c). Two independent strains of rad50-48-PPP are shown. All strains were integrated at their native locus, except rad50-PPP was expressed plasmid based in a rad50Δ strain (marked by red asterisk). Plates were incubated at 30°C. Strains with rad50ΔCC and rad50ΔCC-48 were included as reference. (d) DNA damage sensitivity of the mutants in a mec1Δ strain or a mec1Δ sae2Δ strain towards MMS. (e) Mre11 complex integrity in rad50-PPP “hinge loop” and rad50-48 single and double mutants assessed by co-immunoprecipitation and Western blot with Rad50 or Mre11 antisera.

Supplementary Figure 3 Solution structure of the PfRad50HCC dimer and models for the ‘hook’ and ‘coiled-coil’ mutants.

(a) Molecular envelope of the PfRad50HCC dimer (residues 351-532) calculated from the SAXS data. (b) X-ray scattering profile of PfRad50HCC protein in solution, which was measured at 4°C. The open symbols are the experimental data and the solid line is the X-ray scattering profile. The Guinier plot of PfRad50HCC protein is shown in the inset. (c) Pair distance distribution p(r) function for PfRad50HCC protein, based on an analysis of the experimental SAXS data (empty circles) using the program GNOM56. (d) A model for the SMC hinge mutant. T. maritima SMC hinge domain (residues 475 to 679, magenta and cyan) was fused to HsRad50 coiled-coils (585-651 and 714-766, blue and orange) at N- and C-terminal end, respectively. Detailed construct information is in Supplementary Table 4. (e) A Pfhook mutant dimer model. For FRET analysis, hook domain of PfRad50 (421-476, green and magenta for each monomer) is fused to 651-656 and 715-722 of HsRad50 at N- and C-terminal domain, respectively. (f) A Pfhook monomer model. The hook domain of PfRad50 (421-476, green) is fused to HsRad50 coiled-coils (585-656) and (715 to 766) at N- and C-terminal end, respectively. Gel filtration and ultracentrifuge analyses reveal that this construct favors monomer formation. (g) A model of coiled-coil deletion mutant of Rad50HCC182 (ΔCC mutant). Residues 614-668 and 702-750 are deleted. Residues (585-613, 751-766) of each monomer are shown in light blue and light orange, respectively, and junctions are marked.

Supplementary Figure 4 Gel filtration and circular dichroism analysis of the wild-type and Rad50 apex mutants.

(a) Gel filtration analysis of Rad50HCC182 WT and mutants; Wild-type (blue), SMC hinge (red), Pfhook (green), ΔCC (brown), and ΔCC-48 (magenta). Standard molecular weight markers are marked on top of the graph. The profile for WT Rad50HCC182 in absence of Zn2+ is identical to that in the presence of Zn2+ and omitted for clarity. (b) Circular dichroism analysis of the HsRad50HCC182 WT and “hook” and “coiled-coil” mutants; Wild-type (black), Pfhook (green), ΔCC (red), and ΔCC-48 (blue). Various HsRad50 mutants are monitored by CD spectrophotometer (Jasco J-715) at wavelength of 200-260 nm. We did not examine the SMC hinge mutant because of the large differences in hinge region compared to the hook domain. (c) Analytical ultracentrifuge data of the SMC hinge, Pfhook, ΔCC, and ΔCC-48 (shown from left to right).

Supplementary Figure 5 Telomere lengths in yeast mutants and DNA-binding activities of various HsMRN complexes.

(a) rad50 mutants are defective in telomere maintenance. Telomere length after 0, 15 and 30 generations of growth (0, 1 and 2 days) at 30°C. Heterozygote diploids (RAD50/rad50hook) were included as 0 generations of growth. Genomic DNA was digested with PstI and telomeres were visualized by Southern blot using a telomere specific probe. (b-e) DNA binding activities of various HsMRN complexes. EMSA in the presence of AMPPNP with 0 (lane 1), 100 (lane 2), 200 (lane 3), 400 (lane 4), 600 (lane 5) and 800 nM of WT MRN (lane 6). (b)The WT MRN complex (800 nM) was incubated without AMPPNP in lane 7, (c) MRN with Rad50ΔCC, (d) MRN with Rad50 Pfhook mutant, (e) MRN with Rad50ΔCC-48.

Supplementary Figure 6 Modeling of the yeast rad50-46 coiled-coil-domain suppressor N607Y.

The N607Y suppressor mutant at the coiled-coil of yeast Rad5020 was modeled based on the crystal structure of human Rad50. Two closely located side chains of Tyr607 from the Rad50 dimer may facilitate the coiled-coil interactions between the Rad50 dimer.

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Park, Y., Hohl, M., Padjasek, M. et al. Eukaryotic Rad50 functions as a rod-shaped dimer. Nat Struct Mol Biol 24, 248–257 (2017). https://doi.org/10.1038/nsmb.3369

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