2'-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis

Funding: Engineering and Physical Sciences Research Council [EP/M019195/1]; Engineering and Physical Sciences Research Council Studentship (to J.S.H.); Biotechnology and Biological Sciences Research Council [BB/J001694/2, BB/R021848/1]; ADTBio; University of Kentucky and NCI Cancer Center Support Grant [P30 CA177558]; The Carmen L. Buck Endowment; Emerging Fields Initiative of the Friedrich-Alexander-University of Erlangen-Nuremberg [Grant title ‘Chemistry in Live Cells’]; Wellcome Trust [099149/Z/12/Z]; Royal Society, University Research Fellowship (to J.E.L.). Funding for open access charge: University of Oxford.The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.Publisher PDFPeer reviewe

Unique architecture of thermophilic archaeal virus APBV1 and its genome packaging

Archaeal viruses have evolved to infect hosts often thriving in extreme conditions such as high temperatures. However, there is a paucity of information on archaeal virion structures, genome packaging, and determinants of temperature resistance. The rod-shaped virus APBV1 (Aeropyrum pernix bacilliform virus 1) is among the most thermostable viruses known; it infects a hyperthermophile Aeropyrum pernix, which grows optimally at 90 degrees C. Here we report the structure of APBV1, determined by cryo-electron microscopy at near-atomic resolution. Tight packing of the major virion glycoprotein (VP1) is ensured by extended hydrophobic interfaces, and likely contributes to the extreme thermostability of the helical capsid. The double-stranded DNA is tightly packed in the capsid as a left-handed superhelix and held in place by the interactions with positively charged residues of VP1. The assembly is closed by specific capping structures at either end, which we propose to play a role in DNA packing and delivery.Peer reviewe

The HelQ human DNA repair helicase utilizes a PWI-like domain for DNA loading through interaction with RPA, triggering DNA unwinding by the HelQ helicase core

Genome instability is a characteristic enabling factor for carcinogenesis. HelQ helicase is a component of human DNA maintenance systems that prevent or reverse genome instability arising during DNA replication. Here, we provide details of the molecular mechanisms that underpin HelQ function — its recruitment onto ssDNA through interaction with RPA, and subsequent translocation of HelQ along ssDNA. We describe for the first time a functional role for the non-catalytic N-terminal region of HelQ, by identifying and characterising its PWI-like domain. We present evidence that this domain of HelQ mediates interaction with RPA that orchestrates loading of the helicase domains onto ssDNA. Once HelQ is loaded onto the ssDNA, ATP-Mg2+ binding in the catalytic site activates the helicase core and triggers translocation along ssDNA as a dimer. Furthermore, we identify HelQ-ssDNA interactions that are critical for the translocation mechanism. Our data are novel and detailed insights into the mechanisms of HelQ function relevant for understanding how human cells avoid genome instability provoking cancers, and also how cells can gain resistance to treatments that rely on DNA crosslinking agents

Etudes structurales sur le complexe spécifique de l arn polymérase III C82/C34/C31 et le facteur de transcription végétal Leafy

The first part of my thesis describes my work on the RNA polymerase III specific subcomplex. Since the first studies on the subcomplex C82/C34/C3l there were a number of functional studies on this subcomplex that revealed its involvement in the transcription pre initiation process. During my thesis 1 tried to advance more deeply into the understanding of this process from the structural point of view by X-ray crystallography. Although no crystals of the subcomplex could be obtained, a wealth ofvaluable results have been gathered on different strategies of co-expression of the components of the subcomplex and the crystallization approaches that 1 have explored during my thesis. On the other hand, 1 established the minimal interacting parts of the proteins in the C82/C34/C3l subcomplex and in the TFIIffi complex. One of the most intriguing pro cesses in plant development is the switch from the vegetative into generative growth. LEAFY (LFY) is a key player in this transition as it integrates signaIs from multiple pathways and induces the differentiation of the floral meristem. On the other hand, LFY is found in non-flowering plants, in which it controls the key transitions in the plant life cycle. ln spite of a number of genetics studies on LFY, The function of LFY on the molecular level remained elusive until now. ln collaboration with the group ofF. Parcy (CEA, Grenoble) we solved the crystal structure of the LFY DNA binding domain in complex with two different DNA fragments bearing the sequences from the AP-l and AG-I promoters recognized by LFY. This work is presented in the second part of the thesis.Dans cette thèse deux sujets sont abordés. Dans la première partie de thèse les résultas de la caractérisation structurale du sous-complexe C82/C34/C3l obtenus pendant les deux premières années de ma thèse sont présentés. La plupart des expériences effectuées pendant cette période avaient pour but la production de cristaux du sous-complexe qui auraient servi pour la collection des données de diffraction. Dans ma thèse, je décris différentes approches pour la production du sous-complexe et pour la cristallisation. Malheureusement, les cristaux du sous-complexe n'ont jamais été obtenus. La deuxième partie de mon travail portait sur la caractérisation biochimique et structurale du complexe C82/C34/C31. Une analyse des données biochimiques sur l'interaction entre la sous-unité C34 et des composants du complexe TFIIIB est exposée en détail. Dans la deuxième partie de cette thèse je décris l'étude structurale du facteur de transcription végétal LEAFY par cristallographie aux rayons X. En collaboration avec l'équipe de Dr. François Parcy (CEA, Grenoble), nous avons obtenu la structure de ce facteur de transcription en complexe avec deux fragments d'ADN des promoteurs APl et AG-I avec des résolutions de 2.1 et 2.3Â. Ces structures ont permis d'établir les déterminants majeurs de la reconnaissance spécifique de l'ADN par LFY. Une liaison coopérative des deux monomères de LFY au site APl et AG-I explique en partie la fonction de LFY comme un déclencheur de la transition du méristème vers son état déterminé.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Structural Basis of TBX5–DNA Recognition: The T-Box Domain in Its DNA-Bound and -Unbound Form

International audienc

Dissecting Henipavirus W proteins conformational and fibrillation properties: contribution of their N‐ and C‐terminal constituent domains

International audienceThe Nipah and Hendra viruses are severe human pathogens. In addition to the P protein, their P gene also encodes the V and W proteins that share with P their N‐terminal intrinsically disordered domain (NTD) and possess distinct C‐terminal domains (CTDs). The W protein is a key player in the evasion of the host innate immune response. We previously showed that the W proteins are intrinsically disordered and can form amyloid‐like fibrils. However, structural information on W CTD (CTD W ) and its potential contribution to the fibrillation process is lacking. In this study, we demonstrate that CTD WS are disordered and able to form dimers mediated by disulfide bridges. We also show that the NTD and the CTD W interact with each other and that this interaction triggers both a gain of secondary structure and a chain compaction within the NTD. Finally, despite the lack of intrinsic fibrillogenic properties, we show that the CTD W favors the formation of fibrils by the NTD both in cis and in trans . Altogether, the results herein presented shed light on the molecular mechanisms underlying Henipavirus pathogenesis and may thus contribute to the development of targeted therapies

Functional benefit of structural disorder for the replication of measles, Nipah and Hendra viruses

International audienceAbstract Measles, Nipah and Hendra viruses are severe human pathogens within the Paramyxoviridae family. Their non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the viral RNA-dependent-RNA-polymerase (RpRd) for transcription and replication. The RpRd is a complex made of the large protein (L) and of the phosphoprotein (P), the latter serving as an obligate polymerase cofactor and as a chaperon for N. Both the N and P proteins are enriched in intrinsically disordered regions (IDRs), i.e. regions devoid of stable secondary and tertiary structure. N possesses a C-terminal IDR (NTAIL), while P consists of a large, intrinsically disordered N-terminal domain (NTD) and a C-terminal domain (CTD) encompassing alternating disordered and ordered regions. The V and W proteins, two non-structural proteins that are encoded by the P gene via a mechanism of co-transcriptional edition of the P mRNA, are prevalently disordered too, sharing with P the disordered NTD. They are key players in the evasion of the host antiviral response and were shown to phase separate and to form amyloid-like fibrils in vitro. In this review, we summarize the available information on IDRs within the N, P, V and W proteins from these three model paramyxoviruses and describe their molecular partnership. We discuss the functional benefit of disorder to virus replication in light of the critical role of IDRs in affording promiscuity, multifunctionality, fine regulation of interaction strength, scaffolding functions and in promoting liquid–liquid phase separation and fibrillation

Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins

International audienceHenipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light ofthe critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins

RPA

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo

Redox-dependent formation of a viral amyloid and functional impact

Abstract The Hendra and Nipah viruses (HeV and NiV) are zoonotic biosafety level-4 pathogens within the Paramyxoviridae family. We previously showed that their W proteins form amyloid-like fibrils in vitro . Here, we demonstrate that W also forms fibrils in cellula and that cysteine residues are crucial in dictating the ability of W proteins to fibrillate. The cysteine oxidation state acts as a switch to generate either amorphous aggregates or flexible fibrils. Ectopic expression of W HeV induces an oxidative stress and W HeV fibrils were observed in the nuclei of different cell lines, with fibrillation being impaired by cysteine substitutions. Finally, nuclear fibrils are associated with an impairment of the NF-κB pathway in W HeV transfected cells. This work provides experimental evidence for the ability of Henipavirus W proteins to fibrillate in transfected cells and the first clues on their functional impact. Significance Statement Nipah and Hendra viruses are severe pathogens infecting humans and livestock, classified among the 8 highest priorities for research by the WHO. The W protein, along with the V protein, is a virulence factor responsible for antiviral response inhibition and we demonstrate here that its fibrillation into amyloid-like fibrils occurs in the nucleus of transfected cells, with their formation being dependent of the redox state of the W cysteine residues. The sole transfection of W provokes the production of reactive oxygen species, creating a suitable environment for the fibrils to form. Finally, we show that W fibrils enhance the repression of the antiviral response, thus pointing to W fibrillation as a new promising antiviral target