23 results found
Yates LA, Zhang X, 2023, Phosphoregulation of the checkpoint kinase Mec1ATR, DNA REPAIR, Vol: 129, ISSN: 1568-7864
Liang P, Lister K, Yates L, et al., 2023, Phosphoregulation of DNA repair via the Rad51 auxiliary factor Swi5-Sfr1, Journal of Biological Chemistry, Vol: 299, Pages: 1-15, ISSN: 0021-9258
Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks, the most severe form of DNA damage. The Rad51 protein is central to HR, but multiple auxiliary factors regulate its activity. The heterodimeric Swi5-Sfr1 complex is one such factor. It was previously shown that two sites within the intrinsically disordered domain of Sfr1 are critical for the interaction with Rad51. Here, we show that phosphorylation of five residues within this domain regulates the interaction of Swi5-Sfr1 with Rad51. Biochemical reconstitutions demonstrated that a phosphomimetic mutant version of Swi5-Sfr1 is defective in both the physical and functional interaction with Rad51. This translated to a defect in DNA repair, with the phosphomimetic mutant yeast strain phenocopying the previously established interaction mutant. Interestingly, a strain in which Sfr1 phosphorylation was blocked also displayed sensitivity to DNA damage. Taken together, we propose that controlled phosphorylation of Sfr1 is important for the role of Swi5-Sfr1 in promoting Rad51-dependent DNA repair.
Zhang X, Yates L, Morgan M, 2023, A DNA damage-induced phosphorylation circuit enhances Mec1ATR Ddc2ATRIP recruitment to Replication Protein A, Proceedings of the National Academy of Sciences of USA, Vol: 120, Pages: 1-10, ISSN: 0027-8424
The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1–Ddc2 “senses” single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage–induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2–RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1–Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1–Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1–Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.
Yates LA, 2021, Regulation of DNA break repair by RNA, PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, Vol: 163, Pages: 23-33, ISSN: 0079-6107
Tannous EA, Yates LA, Zhang X, et al., 2021, Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase, Nature Structural and Molecular Biology, Vol: 28, Pages: 50-61, ISSN: 1545-9985
In response to DNA damage or replication fork stalling, the basal activity of Mec1ATR is stimulated in a cell-cycle-dependent manner, leading to cell-cycle arrest and the promotion of DNA repair. Mec1ATR dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo-electron microscopy, we determine the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å and the wild type at 3.8 Å, both in complex with Mg2+-AMP-PNP. These structures yield a near-complete atomic model for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.
Williams R, Yates L, Zhang X, 2020, Structures and regulations of ATM and ATR, master kinases in genome integrity, Current Opinion in Structural Biology, Vol: 61, Pages: 98-105, ISSN: 0959-440X
Homologous recombination (HR) is a faithful repair mechanism for double stranded DNA breaks. Two highly homologous master kinases, the tumour suppressors ATM and ATR (Tel1 and Mec1 in yeast), coordinate cell cycle progression with repair during HR. Despite their importance, our molecular understanding of these apical coordinators has been limited, in part due to their large sizes. With the recent development in cryo-electron microscopy, significant advances have been made in structural characterisation of these proteins in the last two years. These structures, combined with new biochemical studies, now provide a more detailed understanding of how a low basal activity is maintained and how activation may occur. In this review, we summarize recent advances in the structural and molecular understanding of these key components in HR, compare the common and distinct features of these kinases and suggest aspects of structural components that are likely to be involved in regulating its activity.
Yates L, 2020, Being a caregiver while caring about a PhD., Nature
Yates L, Williams R, Hailemariam S, et al., 2020, Cryo-EM structure of nucleotide-bound Tel1ATM unravels the molecular basis of inhibition and structural rationale for disease-associated mutations, Structure, Vol: 28, Pages: 96-104.e3, ISSN: 0969-2126
Yeast Tel1 and its highly conserved human orthologue ATM are large protein kinases centralto the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia(A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Usingcryo electron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Ourstructure reveals molecular details of key residues surrounding the nucleotide binding site andprovides a structural and molecular basis for its intrinsically low basal activity. We show thatthe catalytic residues are in a productive conformation for catalysis, but the PIKK-regulatorydomain-Insert (PRD-I) restricts peptide-substrate access and the N-lobe is in an openconformation, thus explaining the requirement for Tel1 activation. Structural comparisons withother PIKKs suggest a conserved and common allosteric activation mechanism. Our work alsoprovides a structural rationale for many mutations found in A-T and cancer.
Yates LA, Williams RM, Hailemariam S, et al., 2019, Structure of nucleotide-bound Tel1<sup>ATM</sup> reveals the molecular basis of inhibition and structural rationale for disease mutations
<jats:sec><jats:title>SUMMARY</jats:title><jats:p>Yeast Tel1 and its highly conserved human orthologue ATM are large protein kinases central to the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia (A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Using cryo electron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Our structure reveals molecular details of key residues surrounding the nucleotide binding site and provides a structural and molecular basis for its intrinsically low basal activity. We show that the catalytic residues are in a productive conformation for catalysis, but the PIKK-regulatory domain-Insert (PRD-I) restricts peptide-substrate access and the N-lobe is in an open conformation, thus explaining the requirement for Tel1 activation. Structural comparisons with other PIKKs suggest a conserved and common allosteric activation mechanism. Our work also provides a structural rationale for many mutations found in A-T and cancer.</jats:p></jats:sec>
Abdul-Salam V, Russomanno G, Chien-Nien C, et al., 2019, CLIC4/Arf6 pathway – a new lead in BMPRII inhibition in pulmonary hypertension, Circulation Research, Vol: 124, Pages: 52-65, ISSN: 0009-7330
Rationale:Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood.Objective:To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension.Methods and Results:Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium–targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony–forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition.Conclusions:Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.
Yates LA, Aramayo RJ, Pokhrel N, et al., 2018, A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA, Nature Communications, Vol: 9, ISSN: 2041-1723
Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.
Ni T, Kalli AC, Naughton FB, et al., 2017, Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain, BIOCHEMICAL JOURNAL, Vol: 474, Pages: 539-556, ISSN: 0264-6021
Yates LA, Durrant BP, Fleurdepine S, et al., 2015, Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity, NUCLEIC ACIDS RESEARCH, Vol: 43, Pages: 2968-2979, ISSN: 0305-1048
Erskine PT, Fokas A, Muriithi C, et al., 2015, X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure-function studies of a neuronal calcium-signalling protein., Acta Crystallogr D Biol Crystallogr, Vol: 71, Pages: 615-631
The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the pro
Yates LA, Durrant BP, Barber M, et al., 2015, Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1), ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, Vol: 71, Pages: 346-353, ISSN: 2053-230X
Yates LA, Gilbert RJC, 2014, Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
Feng T, Yamamoto A, Wilkins SE, et al., 2014, Optimal Translational Termination Requires C4 Lysyl Hydroxylation of eRF1, MOLECULAR CELL, Vol: 53, Pages: 645-654, ISSN: 1097-2765
Feng T, Yamamoto A, Wilkins SE, et al., 2014, Optimal translational termination requires C4 lysyl hydroxylation of eRF1., Mol Cell, Vol: 53, Pages: 645-654
Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.
Yates LA, Norbury CJ, Gilbert RJC, 2013, The Long and Short of MicroRNA, CELL, Vol: 153, Pages: 516-519, ISSN: 0092-8674
Yates LA, Lumb CN, Brahme NN, et al., 2012, Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 43246-43261
Yates LA, Fuezery AK, Bonet R, et al., 2012, Biophysical Analysis of Kindlin-3 Reveals an Elongated Conformation and Maps Integrin Binding to the Membrane-distal β-Subunit NP<i>X</i>Y Motif, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287
Yates LA, Fleurdepine S, Rissland OS, et al., 2012, Structural basis for the activity of a cytoplasmic RNA terminal uridylyl transferase, NATURE STRUCTURAL & MOLECULAR BIOLOGY, Vol: 19, Pages: 782-787, ISSN: 1545-9993
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