SEARCH · bioRxiv Science

Results for “Biochemistry”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources

bioRxiv2016

Biotinylation by antibody recognition - A novel method for proximity labeling

Identification of protein-protein interactions is a major goal of biological research. Despite technical advances over the last two decades, important but still largely unsolved challenges include the high-throughput detection of interactions directly from primary tissue and the identification of interactors of insoluble proteins that form higher-order structures. We have developed a novel, proximity-based labeling approach that uses antibodies to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues. We used this method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. Our results suggest a considerable variation in the composition of the nuclear envelope of different tissues. Of note, DNA damage response proteins Ku70 and Ku80 are more abundant in the vicinity of lamin A/C after thermal stress. This increased affinity also applies to the progerin isoform, potentially contributing to the premature aging phenotype of Hutchinson-Gilford progeria syndrome. The ability to detect protein-protein interactions in intact tissues, and to compare affinities quantitatively under different conditions or in the presence of disease mutations, can provide a new window into cell biology and disease pathogenesis.

Biochemistry↗

bioRxiv2016

Plant Cysteine Oxidases are Dioxygenases that Directly Enable Arginyl Transferase-Catalyzed Arginylation of N-End Rule Targets

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilisation of group VII ETHYLENE RESPONSE FACTORS (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilisation in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N-terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This is the first molecular evidence showing N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and the first ATE1 substrate in plants. The PCOs and ATE1 may be viable intervention targets to stabilise N-end rule substrates, including ERF-VIIs to enhance submergence tolerance in agronomy.

Biochemistry↗

bioRxiv2016

Molecular mechanisms of substrate-controlled ring dynamics and sub-stepping in a nucleic-acid dependent hexameric motor

Ring-shaped hexameric helicases and translocases support essential DNA, RNA, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the E. coli Rho transcription termination factor as a model system, we have employed solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. SAXS data show that Rho preferentially adopts an open-ring state in solution, and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish the first structural snapshots of substepping events in a hexameric helicase/translocase.\n\nSIGNIFICANCEHexameric, ring-shaped translocases are molecular motors that convert the chemical energy of ATP hydrolysis into the physical movement of protein and nucleic acid substrates. Structural studies of several distinct hexameric translocases have provided insights into how substrates are loaded and translocated; however, the range of structural changes required for coupling ATP turnover to a full cycle of substrate loading and translocation has not been visualized for any one system. Here, we combine low-and high-resolution structural studies of the Rho helicase, defining for the first time the ensemble of conformational transitions required both for substrate loading in solution and for substrate movement by a processive hexameric translocase.

Biochemistry↗

bioRxiv2016

Ligand-induced and small molecule control of substrate loading in a hexameric helicase

Processive, ring-shaped protein and nucleic acid protein translocases control essential biochemical processes throughout biology, and are considered high-prospect therapeutic targets. The E. coli Rho factor is an exemplar hexameric RNA translocase that terminates transcription in bacteria. Like many ring-shaped motor proteins, Rho activity is modulated by a variety of poorly understood mechanisms, including small molecule therapeutics, protein-protein interactions, and the sequence of its translocation substrate. Here, we establish the mechanism of action of two Rho effectors, the antibiotic bicyclomycin and nucleic acids that bind to Rhos primary mRNA recruitment site. Using SAXS and a novel reporter assay to monitor the ability of Rho to switch between open-ring (RNA loading) and closed-ring (RNA translocation) states, bicyclomycin is found to be a direct antagonist of ring closure. Reciprocally, the binding of nucleic acids to its N-terminal RNA recruitment domains is shown to promote the formation of a closed-ring Rho state, with increasing primary site occupancy providing additive stimulatory effects. This study establishes bicyclomycin as a conformational inhibitor of Rho ring dynamics, highlighting the utility of developing assays that read out protein conformation as a prospective screening tool for ring-ATPase inhibitors. Our findings further show that the RNA sequence specificity used for guiding Rho-dependent termination derives in part from an intrinsic ability of the motor to couple the recognition of pyrimidine patterns in nascent transcripts to RNA loading and activity.\n\nSIGNIFICANCEMany processive, ring-ATPase motor proteins rely on substrate-dependent conformational changes to assist with the loading of client substrates into the central pore of the enzyme and subsequent translocation. Using the E. coli Rho transcription terminator as a model hexameric helicase, we show that two distinct ligands - the antibiotic bicyclomycin and pyrimidine-rich nucleic acids - alternatively repress or promote, respectively, the transition of Rho from an open, RNA-loading configuration to a closed-ring, active helicase. Our findings explain several mechanisms by which Rho activity is controlled, and provide a general illustration of how intrinsic and extrinsic factors can regulate ring-type ATPase dynamics through diverse mechanisms.

Biochemistry↗

bioRxiv2016

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

Reactive oxygen species (ROS) have emerged as important cellular signaling agents for survival. Herein, we demonstrate that ROS-mediated oxidation of DNA to yield 8-oxo-7,8-dihydroguanine (OG) in gene promoters is a signaling agent for gene activation. Enhanced gene expression occurs when OG is formed in guanine-rich, potential G-quadruplex sequences (PQS) in promoter coding strands to initiate base excision repair (BER) by 8-oxoguanine DNA glycosylase (OGG1) yielding an abasic site (AP). The AP enables melting of the duplex to unmask the PQS to adopt a G-quadruplex fold in which apurinic/apyrimidinic endonuclease 1 (APE1) binds, but inefficiently cleaves, the AP for activation of VEGF or NTHL1 genes. This concept allowed identification of 61 human DNA repair genes that might be activated by this mechanism. Identification of the oxidatively-modified DNA base OG as guiding protein activity on the genome and altering cellular phenotype ascribes an epigenetic role to OG.

Biochemistry↗

bioRxiv2016

Cooperative DNA binding by a splice variant of the transcription factor ETS1 that lacks autoinhibition

ETS1 is the archetypal member of the metazoan ETS transcription factor family. All the members of this family bind to the conserved GGA(A/T) core ETS binding sequence (EBS). Several ETS family members exhibit autoinhibition, wherein one domain of ETS blocks its own DNA-binding activity until certain conditions are met. Relief of autoinhibition in these proteins is coupled to homo- or hetero-dimer formation. Relief of autoinhibition in full-length ETS1 is catalyzed by non-specific interaction with DNA, which facilitates the formation of protein dimers.The ETS1 splice variant, ETS1-p42, lacks an exon that encodes a crucial part of the autoinhibitory module. Thus,ETS1-p42 does not autoinhibit. We showed that the absence of autoinhibition allows ETS1{Delta}335, an N-terminal deletion that recapitulates the DNA-binding of ETS1-p42, to form homodimers in the absence of DNA. However, ETS1-p42 is thought to bind its DNA sites only as monomers. Here, we explore this paradox and show that ETS1{Delta}335 binds cooperatively to DNA containing two EBSs. We also show that residues in ETS1s DNA recognition helix mediate this DNA-binding cooperativity. Furthermore we also find that a single EBS can bind two ETS1{Delta}335 subunits. Finally, we show that DNA acts catalytically to separate unbound ETS1{Delta}335 dimers into monomers. Together,these results suggest a model of ETS1-p42 DNA-binding, where ETS1-p42 can bind to a single EBS with 2:1 stoichiometry.

Biochemistry↗

bioRxiv2016

CH···O Interactions Are Not the Cause of Trends in Reactivity and Secondary Kinetic Isotope Effects for Enzymatic SN2 Methyl Transfer Reactions

Compaction mattersSN2 substitution represents an important class of reaction for both chemical and biological systems. The ability to assess enzymatic transition state structure within this class of reaction remains a major experimental challenge. Here, we comment on and compare the relative impact of compaction along the axis of reaction to the impact of an orthogonal CH{middle dot}{middle dot}{middle dot}O hydrogen bonding interaction. The latter is concluded to play a limited role in determining relative reaction rates and secondary KIEs derived from experimental structure-activity correlations.

Biochemistry↗

bioRxiv2016

FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

Significance statementSome 25 years ago it was discovered that the activity of the ER chaperone BiP is regulated by covalent modification, the nature of which, AMPylation (not ADPribosylation, as had long been thought) and the enzyme responsible, FICD, have only recently been identified. Genetic inactivation of FICD and in vitro studies of the purified enzyme and substrate have done much to clarify the biochemical consequences of the modification and its underlying logic: As ER stress wanes, FICD uses ATP to AMPylate Thr518 of BiP locking BiP in a relatively inactive conformation. As ER stress levels re-mount the cells draw on this pool of inactive chaperone, which is de-AMPylated and restored to its fully active state.\n\nHere we report on the identity of the de-AMPylating enzyme - and with it on the surprising finding that both AMPylation and de-AMPylation of BiP are carried out by the same polypeptide (FICD) using the same active site, both in vivo and in vitro. Analysis of the reaction products reveals that de-AMPylation does not involve trivial concentration-dependent micro-reversibility of an enzymatic reaction, but rather a switch in the active site of FICD that facilitates two antagonistic thermodynamically favored reactions.\n\nSurprisingly BiP de-AMPylation (not AMPylation) is the default activity of FICD. The side-chain of a single regulatory residue, E234, toggles the enzyme between de-AMPylation and AMPylation in vitro. Our studies thereby uncover an active mechanism that must exist in the ER for coupling waning levels of unfolded protein stress to the conversion of FICD from its default de-AMPylation mode to BiP AMPylation. Whilst the details of this active switch remain to be discovered, we are able to suggest a plausible mechanism by which it may come about.\n\nIdentification of the enzyme that de-modifies BiP to reactivate it will be of interest to cell biologists, whereas the novel features of FICD as a dualfunctioning enzyme with a single bi-functional active site will be of broad interest to enzymologists and molecular biologists.\n\nAbstractProtein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response (UPR) that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoanspecific FIC-domain containing ER-localized enzyme, FICD/HYPE, rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here it is shown that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD-/- cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.

Biochemistry↗

bioRxiv2016

Molecular basis of outer kinetochore assembly on CENP-T

Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.

Biochemistry↗

bioRxiv2016

eIF3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2{middle dot}GTP{middle dot}Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncover a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

Biochemistry↗

bioRxiv2016

A Multi-Crystal Method for Extracting Obscured Signal from Crystallographic Electron Density

Macromolecular crystallography is relied on to reveal subtle atomic difference between samples (e.g. ligand binding); yet their detection and modelling is subjective and ambiguous density is experimentally common, since molecular states of interest are generally only fractionally present. The existing approach relies on careful modelling for maximally accurate maps to make contributions of the minor fractions visible (1); in practice, this is time-consuming and non-objective (2-4). Instead, our PanDDA method automatically reveals clear electron density for only the changed state, even from poor models and inaccurate maps, by subtracting a proportion of the confounding ground state, accurately estimated by averaging many ground state crystals. Changed states are objectively identifiable from statistical distributions of density values; arbitrarily large searches are thus automatable. The method is completely general, implying new best practice for all changed-state studies. Finally, we demonstrate the incompleteness of current atomic models, and the need for new multi-crystal deconvolution paradigms.\n\nOne Sentence SummaryNormally uninterpretable map regions are reliably modelled by deconvoluting superposed crystal states, even with poor starting models.

Biochemistry↗

bioRxiv2016

Identification of quercetin from fruits to immediately fight Zika

Zika virus is spread mainly by the bite of an infected mosquito, which can be passed from a pregnant woman to her fetus, thus leading to birth defects including more than microcephaly. It has been recently estimated that one-third of the world population will be infected by Zika in the near future, but unfortunately so far there is no vaccine or medicine for Zika. In particular, the special concern on the vaccine treatment to Zika and Dengue arising from antibody-dependent enhancement strongly emphasizes the key role of its NS2B-NS3 protease (NS2B-NS3pro) as a target for anti-Zika drug discovery/design due to its absolutely-essential role in viral replication.\n\nIn response to the current global health emergency triggered by the Zika outbreak, we successfully obtained several active forms of Zika NS2B-NS3pro and further attempted to discover its inhibitors from eatable plants and traditional herbal medicines to immediately fight Zika. Here, for the first time, we discovered that quercetin, a flavonoid extensively existing in many fruits and vegetables, effectively inhibits Zika NS2B-NS3pro. We further quantify its inhibitory activity with IC50 of 26.0 {+/-} 0.1 {micro}M; and Ki of 23.0 {+/-} 1.3 {micro}M. As quercetin has been extensively found in fruits, vegetables, leaves and grains, our discovery would benefit the public to immediately fight Zika.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC=\"FIGDIR/small/074559_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (55K):\norg.highwire.dtl.DTLVardef@18e716dorg.highwire.dtl.DTLVardef@b8d5f6org.highwire.dtl.DTLVardef@e7206aorg.highwire.dtl.DTLVardef@11cf2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biochemistry↗

bioRxiv2016

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry

Quantitative proteomics employing mass spectrometry has become an indispensable tool in basic and applied life science research. Methods based on data-dependent acquisition have proved extremely valuable for qualitative proteome analysis but historically have struggled to achieve reproducible quantitative data if large sample cohorts are comparatively analyzed. Targeted proteomics, most commonly implemented as selected reaction monitoring, has emerged as a powerful alternative and succeeded in providing a data independent approach for reproducible quantitative proteomics data but is limited in the number of proteins quantified. SWATH-MS is a recently introduced technique consisting of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, specificity) achieved in targeted proteomics but on the scale of thousands of proteins. While previous SWATH-MS studies have shown high intra-lab reproducibility, this has not been evaluated on an inter-lab basis. In this multi-laboratory evaluation study using data from 11 sites worldwide, we have demonstrated that using SWATH-MS we can consistently detect and quantify more than 4,000 proteins from HEK293 cells and that the quantitative protein data generated across laboratories is reproducible. Using synthetic peptide dilution series, we have shown that the sensitivity, dynamic range and reproducibility established with SWATH-MS methods are also uniformly achieved across labs. This study demonstrates that SWATH-MS is a reproducible and accurate technique that can be confidently deployed for large-scale protein quantification in life science research.

Biochemistry↗

bioRxiv2016

A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT

Bromodomains are critical components of many chromatin modifying/remodeling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. We find that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a novel bromodomain-DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity, specificity, and ability to localize to and compact acetylated chromatin. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify new structural features of BET bromodomains that may be targeted for therapeutic inhibition.

Biochemistry↗

bioRxiv2016

Proper modelling of ligand binding requires an ensemble of bound and unbound states

SynopsisWe emphasise and demonstrate the importance of modelling the superpositions of ligand-bound and unbound states that commonly occur in crystallographic datasets. Generation of an ensemble that describes not only the dominant state in the crystal is important for the high-quality refinement of low-occupancy ligands, as well as to present a model that explains all of the observed density.\n\nAbstractSmall molecules bind to only a fraction of the proteins in the crystal lattice, but occupancy refinement of ligands is often avoided by convention; occupancies are set to unity, assuming that the error will be adequately modelled by the B-factors, and weak ligand density is generally ignored or attributed to disorder. Where occupancy refinement is performed, the superposed atomic state is rarely modelled. We show here that these modelling approaches lead to a degradation of the quality of the ligand model, and potentially affect the interpretation of the interactions between the bound ligand and the protein. Instead, superior accuracy is achieved by modelling the ligand as partially occupied and superposed on a ligand-free \"ground-state\" solvent model. Explicit modelling of the superposed unbound fraction of the crystal using a reference dataset allows constrained refinement of the occupancy of the ligand with minimal fear of over-fitting. Better representation of the crystal also leads to more meaningful refined atomic parameters such as the B-factor, allowing more insight into dynamics in the crystal. We present a simple approach and simple guidelines for generating the ensemble of bound and unbound states, assuming that datasets representing the unbound states (the ground state) are available. Judged by various electron density metrics, ensemble models are consistently better than corresponding single-state models. Furthermore, local modelling of the superposed ground state is found to be generally more important for the quality of the ligand model than convergence of the overall phases.

Biochemistry↗

bioRxiv2016

SCFSlmb recognizes a conserved degron within the survival motor neuron (SMN) self-interaction domain to mediate ubiquitylation of SMN and SMNΔ7 isoforms

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMN{Delta}7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of SMN isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phospho-degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMN{Delta}7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMN{Delta}7S270A, but not wild-type SMN{Delta}7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric, and sequestered when SMN forms higher-order multimers.

Biochemistry↗

bioRxiv2016

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: Conservation of the lateral RNA-binding mode

SynopsisThe structure of an Hfq homolog from the deep-branching thermophilic bacterium Aquifex aeolicus, determined to 1.5-[A] resolution both in apo form and bound to a uridine-rich RNA, reveals a conserved, pre-organized RNA-binding pocket on the lateral rim of the Hfq hexamer.\n\nAbstractThe host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNA) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings, with at least two distinct surfaces that bind RNA. Recently, another binding site--dubbed the lateral rim--has been implicated in sRNA*mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structures and functions of Hfq from basal bacterial lineages such as the Aquificae. Thus, we have cloned, overexpressed, purified, crystallized, and biochemically characterized Aae Hfq. We have determined the structures of Aae Hfq in space-groups P1 and P6, both to 1.5 [A] resolution, and we have discovered nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well-defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

Biochemistry↗

bioRxiv2016

The Chd1 chromatin remodeler shifts hexasomes unidirectionally

Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can be used to produce hexasomes in a specific orientation on DNA, which provide a useful tool for interrogating chromatin enzymes and allow for the generation of precisely defined asymmetry in nucleosomes. Using this methodology, we demonstrate that the Chd1 chromatin remodeler requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.

Biochemistry↗