Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair

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Ctp1 (also known as CtIP or Sae2) collaborates with Mre11–Rad50–Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.

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Figure 1: Tetrameric Ctp1 is an intrinsically disordered protein.
Figure 2: X-ray crystal structure of the Ctp1 N-terminal tetramerization domain.
Figure 3: Ctp1 binds DNA.
Figure 4: Identifying Ctp1 DNA-interaction motifs.
Figure 5: Ctp1 bridges DNA.
Figure 6: Sensitivity of Ctp1 THDD and RHR mutations to DNA-damaging agents.
Figure 7: Effects of Ctp1 THDD and RHR mutations on S. pombe DSB repair.
Figure 8: Overall model for Ctp1 molecular architecture from X-ray structures, SAXS and biophysical analysis.

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Our studies are supported by the US National Institute of Health Intramural Program, US National Institute of Environmental Health Sciences (NIEHS) grants 1Z01ES102765 (R.S.W.) and 1Z01ES021016 (M.A.R.). We thank L. Pedersen of the NIEHS Collaborative crystallography group and the Advanced Photon Source (APS) Southeast Regional Collaborative Access Team (SER-CAT) for beamline access. Use of the APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. We thank M. Junop (University of Western Ontario) for nicked plasmid substrate, J. Williams of the NIEHS protein microcharacterization core for MS analysis, R. Dutcher (NIEHS) for help with MALS analysis and G. Mueller (NIEHS) and B. Wallace (NIEHS) for comments on the manuscript.

Author information

S.N.A., R.S.W., M.A.R., J.W. and J.S.W. designed the experiments. S.N.A. and P.D.R. performed crystallization experiments. S.N.A. and R.S.W. solved and refined the Ctp1 X-ray structure. S.N.A. and R.S.W. carried out SAXS experiments. S.N.A., Y.N. and C.D.A. performed biochemical experiments. S.N.A., J.W., C.D.A. and J.S.W. performed S. pombe experiments. R.S.W. and S.N.A. wrote the manuscript with input from all authors. R.S.W. managed the project.

Correspondence to R Scott Williams.

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Integrated supplementary information

Supplementary Figure 1 Ctp1 small-angle X-ray scattering (SAXS).

SAXS profiles of full length Ctp1 (blue), MBP-Ctp11­–60 (green) and MBP-Ctp115–60 (black). Inset: Guinier plots, colored as for scattering profiles, indicating the absence of aggregated protein in SAXS samples.

Supplementary Figure 2 A hydrophobic core stabilizes the Ctp1 tetrameric helical dimer-of-dimers (THDD) domain.

(a) Electrostatic surface potential representation illustrating the Ctp1 tetramer hydrophobic core and hydrophilic exterior. (b) Helical wheel diagram of Ctp1 parallel dimeric coiled-coil region. The heptad repeat with additional salt bridges (dotted lines) between coils is shown. Amino acid positions are numbered for “a” and “d” positions of the heptad repeat.

Supplementary Figure 3 Experimental electron density of the Ctp1 THDD domain.

A model phased, σ-A weighted 2Fo-Fc electron density map (blue, contoured at 1.0σ) was calculated from the initial molecular replacement polyalanine model solution. The corresponding σ-A weighted Fo-Fc map (green, contoured at 2.0σ) shows positive difference density for unmodeled helical regions, and amino acid side-chains.

Supplementary Figure 4 Oligomerization states of Ctp1 truncations and mutants.

(a) SEC-MALS traces of differential refractive index and molar mass for Ctp161–294 (blue) and Ctp1 THDD mutation (R32A K41A) (red). (b) SEC-MALS traces of differential refractive index and molar mass for N-terminal MBP-tagged Ctp11–60 (green) (as in Fig. 1d) and MBP-tagged Ctp11–60 (H11A W12A Y16A) (brown).

Supplementary Figure 5 Ctp1 binds DNA.

(a) Quantification of Ctp1–DNA binding. Mean values shown. Error bars, s.d. (n=3). (b) Ctp1FL binding variable lengths of double-stranded DNA. Arrow indicates 40­–50bp. (c) Purified Ctp1 deletion and internal deletion protein constructs. (d) Ctp1FL and Ctp1 protein halves binding DNA. (e) Ctp1FL and Ctp1 internal deletion DNA binding assays. WT, wildtype. Ctp1 concentrations expressed as tetrameric (tet) or monomeric (mono) as labeled. Experiments were repeated three times for (a), (b), (d), (e), with representative gels shown.

Supplementary Figure 6 Purified Ctp1FL N- and C-terminal mutations.

(a) Ctp1 N-terminal THDD domain point mutations used in DNA binding studies. (b) Ctp1 C-terminal point mutations used in DNA binding studies. WT, wildtype.

Supplementary Figure 7 Titration of DNA binding by Ctp1 mutants of the conserved RHR motif.

Ctp1 concentrations expressed as tetrameric (tet). Experiment was repeated 3 times with representative gels shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–4 (PDF 3777 kb)

Supplementary Data Set 1

Uncropped gels and blots (PDF 2633 kb)

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Andres, S., Appel, C., Westmoreland, J. et al. Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair. Nat Struct Mol Biol 22, 158–166 (2015) doi:10.1038/nsmb.2945

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