When two is not enough: a CtIP tetramer is required for DNA repair by Homologous Recombination

Forment, Josep V.; Jackson, Stephen D.; Pellegrini, Luca

doi:10.1080/19491034.2015.1086050

Cited by 9 publications

(9 citation statements)

References 35 publications

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“…Indeed, direct observation of purified CtIP using negative stain electron microscopy revealed an extraordinary dumbbell-like structure in which polar globular domains are held about 30 nm apart by a central ‘rod’ (Figure 1B, β class; Figure 1—figure supplement 1). The length of the rod is broadly consistent with the predicted length of the coiled-coil domains, which suggests that the centre of the dumbbell is the site of the tetramerization interface as proposed in a previous model (Forment et al, 2015). In addition to the dumbbell structures, we less frequently observed structures that we call ‘tadpoles’ containing a single globular domain and a tail, which presumably represent a dimeric form of the protein formed during EM sample preparation and deposition (Figure 1B, α class; Figure 1—figure supplement 1).…”

Section: Resultssupporting

confidence: 86%

“…The purified protein is tetrameric and adopts a striking ‘dumbbell’ architecture, which we have observed with both negative-stain EM and AFM. This shape is expected based on the hypothesis that CtIP adopts a ‘dimer of dimers’ arrangement, in which the tips of long N-terminal parallel coiled coils interact to form the tetrameric interface observed in crystal structures (Andres et al, 2015; Davies et al, 2015; Forment et al, 2015) (Figure 7). We found that CtIP bound tightly to DNA using both EMSA and fluorescence anisotropy assays.…”

Section: Discussionmentioning

confidence: 70%

“…A mutation (L27E) that prevented tetramerization also rendered CtIP non-functional in vivo. The proposed model that arises from these reports is one where two rigid coiled coils (aa1-145) protrude in opposite directions from the tetramerisation domain, spanning a distance of about 30 nm, with the unstructured C-termini presumably placed at the opposing ends (Forment et al, 2015). It has been shown that CtIP binds to DNA; for S. pombe Ctp1 a number of DNA substrates have been interrogated by EMSA and were all shown to be bound with similar affinities (Andres et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair

Wilkinson

Martín-González

Kang

et al. 2019

eLife

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CtIP is involved in the resection of broken DNA during the S and G2 phases of the cell cycle for repair by recombination. Acting with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with complex structures including forked ssDNA overhangs and nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

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Section: Resultssupporting

confidence: 86%

Section: Discussionmentioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

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CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair

Wilkinson

Martín-González

Kang

et al. 2019

eLife

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“…Consistent with a strand coordination activity, the architecture of Ctp1 appears appropriate to mediate DNA bridging (Fig. 4B) [5,44,80]. In vitro , purified Ctp1 can link DNA molecules in bridging reactions [5].…”

Section: Ctip Dna Transactionsmentioning

confidence: 88%

CtIP/Ctp1/Sae2, molecular form fit for function

Andres¹,

Williams²

2017

DNA Repair

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Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By modulating the nucleolytic 5′-3′ resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein-protein interactions and DNA binding.

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“…These diverse functions explain the presence of coiled-coils within 10% of eukaryotic proteins (Liu & Rost, 2001), and underlie the importance of their structure elucidation. Further, the geometry of coiled-coils means that the most basic understanding of their structure, namely oligomer state and their parallel or antiparallel orientation, can dramatically transform our understanding of the topology of their wider biological assemblies, such as when they mediate head-to-head association between functional domains (Davies et al, 2015;Forment et al, 2015). Thus, structure solution of coiled-coil proteins is fundamental to our understanding of a wide range of cellular functions.…”

Section: Introductionmentioning

confidence: 99%

Extending the scope of coiled-coil crystal structure solution byAMPLEthrough improvedab initiomodelling

Thomas

Keegan

Rigden

et al. 2020

Acta Crystallogr D Struct Biol

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The phase problem remains a major barrier to overcome in protein structure solution by X‐ray crystallography. In recent years, new molecular‐replacement approaches using ab initio models and ideal secondary‐structure components have greatly contributed to the solution of novel structures in the absence of clear homologues in the PDB or experimental phasing information. This has been particularly successful for highly α‐helical structures, and especially coiled‐coils, in which the relatively rigid α‐helices provide very useful molecular‐replacement fragments. This has been seen within the program AMPLE, which uses clustered and truncated ensembles of numerous ab initio models in structure solution, and is already accomplished for α‐helical and coiled‐coil structures. Here, an expansion in the scope of coiled‐coil structure solution by AMPLE is reported, which has been achieved through general improvements in the pipeline, the removal of tNCS correction in molecular replacement and two improved methods for ab initio modelling. Of the latter improvements, enforcing the modelling of elongated helices overcame the bias towards globular folds and provided a rapid method (equivalent to the time requirements of the existing modelling procedures in AMPLE) for enhanced solution. Further, the modelling of two‐, three‐ and four‐helical oligomeric coiled‐coils, and the use of full/partial oligomers in molecular replacement, provided additional success in difficult and lower resolution cases. Together, these approaches have enabled the solution of a number of parallel/antiparallel dimeric, trimeric and tetrameric coiled‐coils at resolutions as low as 3.3 Å, and have thus overcome previous limitations in AMPLE and provided a new functionality in coiled‐coil structure solution at lower resolutions. These new approaches have been incorporated into a new release of AMPLE in which automated elongated monomer and oligomer modelling may be activated by selecting `coiled‐coil' mode.

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When two is not enough: a CtIP tetramer is required for DNA repair by Homologous Recombination

Cited by 9 publications

References 35 publications

CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair

CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair

CtIP/Ctp1/Sae2, molecular form fit for function

Extending the scope of coiled-coil crystal structure solution byAMPLEthrough improvedab initiomodelling

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