PLK1 regulates CtIP and DNA2 interplay in long-range DNA end resection

Ceppi, Ilaria; Cannavó, Elda; Bret, Hélène; Camarillo, Rosa; Vivalda, Francesca; Thakur, Roshan Singh; Romero-Franco, Amador; Sartori, Alessandro A.; Huertas, Pablo; Guérois, Raphaël; Ćejka, Petr

doi:10.1101/gad.349981.122

Cited by 4 publications

(15 citation statements)

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“…Therefore, although this region may contribute to the overall stability of the DNA interaction either directly or indirectly, it appears insufficient alone to facilitate tight binding. Primary structure analysis and current folding algorithms fail to provide confident insights into CtIP structure beyond the N-terminal coiled-coils (for which models are already available from crystallography) or very small regions of the C-terminus (45, 46). This probably reflects the extensive disorder that is predicted in C-terminal regions of this enigmatic protein.…”

Section: Discussionmentioning

confidence: 99%

“…Primary structure analysis and current folding algorithms fail to provide confident insights into CtIP structure beyond the N-terminal coiled-coils (for which models are already available from crystallography) or very small regions of the C-terminus (45,46). This probably reflects the extensive disorder that is predicted in C-terminal regions of this enigmatic protein.…”

Section: Discussionmentioning

confidence: 99%

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DNA binding and bridging by human CtIP in the healthy and diseased states

Balaji,

De Bragança,

Balaguer-Pérez

et al. 2023

Preprint

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The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed.Significance StatementCtIP helps to repair broken chromosomes through its ability to bind and bridge DNA molecules. We studied the structural and biochemical basis for these activities and how they are affected by hereditary CtIP mutations associated with developmental disorders. We discovered a minimal domain in the C-terminal region of CtIP which supports DNA binding as a monomer. DNA binding is non-specific and facilitates 1D diffusion, but binding alone is insufficient for intermolecular tethering of DNA molecules which requires tetramerization of CtIP via N-terminal coiled-coil domains. All disease variants tested displayed impaired DNA bridging activity. These results have important implications for understanding the role of CtIP as a hub protein for DNA break repair and its dysfunction in human disease.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

DNA binding and bridging by human CtIP in the healthy and diseased states

Balaji,

De Bragança,

Balaguer-Pérez

et al. 2023

Preprint

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“…10 Ceppi et al showed how AF2 models can be used to interpret the functional effect of mutations. 11 Contact maps can also be used to explore the dynamics of AF2 models. Fakhoury et al used contact maps to go beyond the folded state and instead study the folding pathway.…”

Section: Pipelinesmentioning

confidence: 99%

“…Until now, and besides the case of mitofusins that is discussed in the second part of the manuscript, few studies have been conducted with such mutations in mind; for example, Pyatnitskaya et al explored single point mutations that disrupt interactions found on models generated with AlphaFold-Multimer and are associated with functional defects . Ceppi et al showed how AF2 models can be used to interpret the functional effect of mutations …”

Section: Integrating Af2 Into Experimental Pipelinesmentioning

confidence: 99%

A Perspective on the Prospective Use of AI in Protein Structure Prediction

Versini,

Sritharan,

Aykac Fas

et al. 2023

J. Chem. Inf. Model.

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AlphaFold2 (AF2) and RoseTTaFold (RF) have revolutionized structural biology, serving as highly reliable and effective methods for predicting protein structures. This article explores their impact and limitations, focusing on their integration into experimental pipelines and their application in diverse protein classes, including membrane proteins, intrinsically disordered proteins (IDPs), and oligomers. In experimental pipelines, AF2 models help X-ray crystallography in resolving the phase problem, while complementarity with mass spectrometry and NMR data enhances structure determination and protein flexibility prediction. Predicting the structure of membrane proteins remains challenging for both AF2 and RF due to difficulties in capturing conformational ensembles and interactions with the membrane. Improvements in incorporating membrane-specific features and predicting the structural effect of mutations are crucial. For intrinsically disordered proteins, AF2's confidence score (pLDDT) serves as a competitive disorder predictor, but integrative approaches including molecular dynamics (MD) simulations or hydrophobic cluster analyses are advocated for accurate dynamics representation. AF2 and RF show promising results for oligomeric models, outperforming traditional docking methods, with AlphaFold-Multimer showing improved performance. However, some caveats remain in particular for membrane proteins. Real-life examples demonstrate AF2's predictive capabilities in unknown protein structures, but models should be evaluated for their agreement with experimental data. Furthermore, AF2 models can be used complementarily with MD simulations. In this Perspective, we propose a "wish list" for improving deep-learning-based protein folding prediction models, including using experimental data as constraints and modifying models with binding partners or post-translational modifications. Additionally, a meta-tool for ranking and suggesting composite models is suggested, driving future advancements in this rapidly evolving field.

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“…While EXO1 acts on double-stranded DNA (dsDNA), DNA2 nuclease is single-stranded DNA (ssDNA) specific, and thus requires a RecQ family helicase partner, Bloom (BLM) or Werner (WRN), together with the ssDNA binding protein replication protein A (RPA) 1,29 . However, a significant crosstalk exists between the resection pathways 28,[30][31][32][33] . BRCA1-BARD1 promotes resection, as well as the downstream steps in the HR pathway, including RAD51 loading and strand invasion, likely…”

mentioning

confidence: 99%

Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection

Cejka,

Ceppi,

Stritto

et al. 2024

Preprint

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DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by DNA end resection, a process involving the controlled degradation of the 5'-terminated strands at DSB sites 1,2. The breast cancer suppressor BRCA1-BARD1 not only promotes resection and HR, but it also protects DNA upon replication stress 1,3-9. While BRCA1-BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, its direct role in resection has been unclear, particularly due to complex phenotypes associated with the depletion of this essential and multi-functional complex 10-18. Using purified recombinant proteins, we show here that BRCA1-BARD1 directly promotes long-range DNA end resection pathways catalyzed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA unwinding by the WRN/BLM RecQ family helicase. Together with MRE11-RAD50-NBS1 (MRN) and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex 19,20, which stimulates resection synergistically to even a greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1-C complex 21-23, inhibits resection, showing that BRCA1-C is a functionally integrated ensemble directly promoting long-range resection. While BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves an HR-independent function of the recombinase RAD51 4-6,8. We show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA degradation. Based on our data, the presence and local concentration of RAD51 might determine the balance between the pro-nuclease and the DNA protection functions of BRCA1-BARD1 in the various physiological contexts.

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PLK1 regulates CtIP and DNA2 interplay in long-range DNA end resection

Cited by 4 publications

References 72 publications

DNA binding and bridging by human CtIP in the healthy and diseased states

DNA binding and bridging by human CtIP in the healthy and diseased states

A Perspective on the Prospective Use of AI in Protein Structure Prediction

Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection

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