CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair
- University of Bristol, United Kingdom
- Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Spain
- Imperial College London, United Kingdom
Figures
Figure 1 with 4 supplements
Wild type CtIP is a tetrameric protein that forms a dumbbell-shaped particle.
(A) SDS-PAGE and SEC-MALS analysis of wild type CtIP as prepared (lane 1, black trace) and following dephosphorylation post-purification (lane 2, grey trace). Horizontal lines on the SEC-MALS graph …
Figure 1—figure supplement 1
Negative stain electron microscopy.
(A) An example of a micrograph obtained with negative staining of CtIP at 42,000 X magnification. (B) Examples of individual CtIP particles. (C) Further examples of 2D classes for CtIP particles …
Figure 1—figure supplement 2
Further examples and structural interpretation of AFM particle classes.
Class V particles are thought to represent tetrameric CtIP (see main text and Figure 1D for justification), consisting of a dimer-of-dimers arrangement (blue and red) of parallel coiled coils …
Figure 1—figure supplement 3
The rod between the globular domains is flexible and variable in length.
(A) Examples of CtIP particles displaying significantly different intervening lengths between spots. (B) The distribution of the distances between bright spots in CtIP particles is centred at ~ 30 …
Figure 1—figure supplement 4
CtIP purified from insect cells is hyper-phosphorylated.
Human wild type CtIP was expressed in insect cells, purified in the presence of dephosphorylase inhibitors, and analysed for post-translational modifications by Orbitrap LC-MS/MS mass spectrometry. …
Figure 2
Mutation in the N-terminal coiled-coil domain prevents CtIP tetramerisation.
(A) SDS-PAGE and SEC-MALS analysis of CtIP L27E (lane 1, blue trace) and CtIP R839A (lane 2, red trace). Data for wild type CtIP is also shown for comparison (dotted black lines). (B) AFM imaging of …
Figure 3
CtIP binds preferentially to ss-dsDNA Y-junctions in a manner dependent on both the N-terminal tetramerisation and C-terminal DNA binding motifs.
(A) Electrophoretic mobility shift assay. Radiolabelled DNA molecules with the different structures (indicated) were incubated with increasing concentrations of CtIP tetramer and run on …
Figure 4 with 1 supplement
Dephosphorylation of CtIP potentiates DNA binding and facilitates determination of the DNA binding stoichiometry.
(A) EMSA assays comparing the binding of wild type CtIP as prepared and following treatment with λ phosphatase (denoted CtIPλ) as described in the Materials and methods. (B) Fluorescence anisotropy …
Figure 4—figure supplement 1
Real time measurement of DNA binding potentiation upon treatment of CtIP with λ phosphatase.
(A) Principle of the assay. CtIP (blue) was added to 5 nM HEX-labelled fork DNA (purple) at a concentration equal to ~ 0.5 x Kd resulting in a low fluorescence anisotropy reading (~0.13). A small …
Figure 5 with 4 supplements
CtIP binding to DNA is stabilised by DNA Y-junctions and DNA end blocks.
(A) Principle of the competition DNA unbinding assay monitored by fluorescence anisotropy. HEX-labelled and unlabelled DNA fork molecules are shown in purple and black respectively. CtIP is in blue. …
Figure 5—figure supplement 1
Structures of competitor DNA molecules used in this study.
The substrates are numbered as in Supplementary file 1. The arrows represent the 3′-ends of DNA, a yellow circle indicates biotin and the purple square is streptavidin. The blue numbers represent …
Figure 5—figure supplement 2
Raw data for all IC50 measurements.
Numbers above each graph indicate the competitor DNA used as per Supplementary file 1.
Figure 5—figure supplement 3
IC50 values for CtIP binding measured for a wider range of competitor DNA molecules.
(A) Comparison of IC50 values for ssDNA molecules of different length as indicated (B) Comparison of how streptavidin binding affects IC50 values for duplex DNA molecules containing different …
Figure 5—figure supplement 4
CtIP and Ku display distinctive DNA binding modes.
(A) Principle of experiment to compare the effects of DNA end blocks on DNA binding by CtIP and Ku. CtIP and Ku are pre-bound at 2xKd to HEX-labelled probe DNA (a fork for CtIP and an oligoduplex …
Figure 6 with 4 supplements
CtIP promotes intermolecular DNA bridging.
(A) Representative AFM images of forked DNA substrates in the absence of CtIP. The contour length histogram shows a single gaussian peak centred on a value equivalent to a single contour length. (B) …
Figure 6—figure supplement 1
Evidence for CtIP bound to DNA at bridging interfaces.
(A) An example of a bridged DNA molecule bound to CtIP obtained at a 1:19 DNA:CtIP4 ratio (B) Height profile along the molecule of interest. Section A is interpreted as DNA (188 nm contour length), …
Figure 6—figure supplement 2
CtIP promotes intramolecular DNA bridging.
(A) Example of an AFM image showing an internally-bridged (circularised) DNA molecule. (B) Height profile for the boxed molecule shown in A reveals additional height associated with the …
Figure 6—figure supplement 3
Assessment of nuclease activity in CtIP preparations.
It is currently controversial as to whether purified CtIP and its orthologues possess intrinsic nuclease activity (Andres and Williams, 2017). Therefore, to test our CtIP preparations for nuclease …
Figure 6—figure supplement 4
Dephosphorylated CtIP promotes intermolecular DNA bridging.
(A) Representative AFM images of forked DNA substrates in the absence of dephosphorylated CtIP (CtIPλ). The contour length histogram shows a single gaussian peak centred on a value equivalent to a …
Figure 7
A speculative model for binding and bridging of broken DNA ends by CtIP.
The CtIP monomer comprises at least three functional regions; an N-terminal tetramerization domain, a central region of predicted disorder, and a C-terminal DNA binding domain. This assembles to …
Author response image 1
Moderately elevated MgCl2 concentrations dramatically reduce the affinity of CtIP for DNA.
https://doi.org/10.7554/eLife.42129.025Additional files
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Supplementary file 1
Comprehensive data for DNA competition assay and details of substrate construction.
Supplementary Table 1: IC50 values for competitor DNA molecules used in DNA unbinding assays. The reported error is the error associated with the fit to a hyperbolic unbinding curve as shown in Figure 5—figure supplement 2. Supplementary Table 2: Assembly/Source of DNA substrates. Small DNA substrates were prepared by annealing different combinations of short oligonucleotides (A-T). The sequences for the oligonucleotides are presented in Supplementary Tables 3. Supplementary Table 3: Sequences of oligonucleotides used to assemble competitor DNA molecules.
- https://doi.org/10.7554/eLife.42129.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.42129.023
