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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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.
The authors declare no competing financial interests.
Integrated supplementary information
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.
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.
(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).
(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.
(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.
Ctp1 concentrations expressed as tetrameric (tet). Experiment was repeated 3 times with representative gels shown.
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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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