DNA Wrapping Research Articles

Thermodynamics of E. coli SSB phase separation. Effects of monovalent salt concentration and type and intrinsically disordered linker modifications.

E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. Depending on solution conditions SSB can bind to ssDNA in different binding modes characterized by different extent of DNA wrapping around the SSB tetramer and different cooperativities. We and others showed recently that SSB can undergo salt dependent phase separation (PS) which is facilitated by increases in concentration of the physiological salt, KGlu, while inhibited by [KCl] and/or deletion of the intrinsically disordered linker ( IDL) within the SSB C-terminal tail. These same factors influence non-nearest neighbor (NNN) cooperative binding of SSB to long ssDNA resulting in collapse of the nucleoprotein complexes. Here we report a thermodynamic study of EcSSB PS over a broad range of [KCl] and [KGlu] and find that PS is driven by favorable enthalpy with unfavorable entropy changes. While the effects of [KGlu] and [KCl] are similar at low salt concentrations, dramatic differences are observed at [salt] above 0.1 M. At low [salt] PS is driven mostly by electrostatic (coulombic) interactions, whereas at high [salt] Hofmeister effects dominate involving weak preferential interactions relative to water. We also have examined the effects on PS of amino acid sequence and composition of the IDL. The results are discussed in relation with SSB function in E. coli (supported by NIH GM136632 to TML and GM118100 to MTR).

Cryo–electron microscopy structure of the H3-H4 octasome: A nucleosome-like particle without histones H2A and H2B

The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo-electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4' four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone-histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.

Free Energy Landscape of H2A-H2B Displacement From Nucleosome

Nucleosome reconstitution plays an important role in many cellular functions. As an initial step, H2A-H2B dimer displacement, which is accompanied by disruption of many of the interactions within the nucleosome, should occur. To understand how H2A-H2B dimer displacement occurs, an adaptively biased molecular dynamics (ABMD) simulation was carried out to generate a variety of displacements of the H2A-H2B dimer from the fully wrapped to partially unwrapped nucleosome structures. With regards to these structures, the free energy landscape of the dimer displacement was investigated using umbrella sampling simulations. We found that the main contributors to the free energy were the docking domain of H2A and the C-terminal of H4. There were various paths for the dimer displacement which were dependent on the extent of nucleosomal DNA wrapping, suggesting that modulation of the intra-nucleosomal interaction by external factors such as histone chaperones could control the path for the H2A-H2B dimer displacement. Key residues which contributed to the free energy have also been reported to be involved in the mutations and posttranslational modifications (PTMs) which are important for assembling and/or reassembling the nucleosome at the molecular level and are found in cancer cells at the phenotypic level. Our results give insight into how the H2A-H2B dimer displacement proceeds along various paths according to different interactions within the nucleosome.

The Effects of Site Specific Histone Modifications on Nucleosomal DNA Accessibility

Chromatin accessibility is the ability of nucleosomal DNA to be physically accessed by cellular biomachinery. Accessibility of chromatin is controlled by a slew of post translational modifications to histone proteins, DNA, and chromatin‐binding factors. This growing field of research has become increasingly important as chromatin accessibility has a large effect on transcription and gene regulation. Gene and cell cycle regulation must be understood to properly study their misregulation which commonly occurs in most, if not all, cancers. Thus, understanding the organization and regulation of the chromatin state by post translational modifications plays an important role in understanding the dysfunction of cancerous cells. The most basic unit of chromatin is the nucleosome core particle. The nucleosome core particle is made up of an octamer that has two copies each of the 4 histone proteins H2A, H2B, H3, and H4 with DNA wrapping around the octamer itself. It is on the N‐terminal tails of these histone proteins that many post‐translational modifications take place the regulate the chromatin state. In this study, the effect of histone lysine acetylation on the DNA‐histone core interaction will be assessed by its thermal stability and accessibility of nucleosomal DNA to Pst1 Digestion. We propose to assay singly acetylated mononucleosomes at the following sites: H2A: K5; H2B: K5, K12, K15, and K20; H3: K9, K14, K18, K23, K27, K36, K37, K64, K56, K79, K115, and K122; H4: K5, K8, K12, and K16. We predict that, as previously shown with nu′‐H3K18ac and nu′‐H3K36ac acetylation at lysine sites will facilitate unwrapping of nucleosomal DNA.

Potential of Mean Force for DNA Wrapping Around a Cationic Nanoparticle.

Sharp bending and wrapping of DNA around proteins and nanoparticles (NPs) has been of extensive research interest. Here, we present the potential of mean force (PMF) for wrapping a DNA double helix around a cationic NP using coarse-grained models of a double-stranded DNA and a cationic NP. Starting from a NP wrapped around by DNA, the PMF was calculated along the distance between the center of the NP and one end of the DNA molecule. A relationship between the distance and the extent of DNA wrapping is used to calculate the PMF as a function of DNA wrapping around a NP. In particular, the PMF was compared for two DNA sequences of (AT)25/(AT)25 and (AC)25/(GT)25, for which the persistence lengths are different by ∼10 nm. The simulation results provide solid evidence of the thermodynamic preference for complex formation of a cationic NP with more flexible DNA over the less flexible DNA. Furthermore, we estimated the elastic energy of DNA bending, which was in good order-of-magnitude agreement with the theoretical prediction of elastic rods. This work suggests that the variation of sequence-dependent DNA flexibility can be utilized in DNA nanotechnologies, in which the position and dynamics of NPs are regulated on large-scale DNA structures, or the structural transformation of DNA is triggered by the sequence-dependent binding of NPs.

Integrative Chemical Biology Approaches to Deciphering the Histone Code: A Problem-Driven Journey.

The hereditary blueprint of a eukaryotic cell is encoded in its genomic DNA that is tightly compacted into chromatin together with histone proteins. The basic repeating units of chromatin fibers are nucleosomes, in which approximately 1.7 turns of DNA wrap around a proteinaceous octamer consisting of two copies of histones H2A, H2B, H3, and H4. Histones are extensively decorated by a variety of posttranslational modifications (PTMs, e.g., methylation, acetylation, ubiquitylation, phosphorylation, etc.), serving as one of the cellular mechanisms that regulates DNA-templated processes, including but not limited to gene transcription, DNA replication, and DNA damage repair. Most of the histone PTMs exist in dynamic fluctuations, and their on and off states are exquisitely regulated by enzymes known as "writers" and "erasers", respectively. When installed at certain sites, histone PTMs can change the local physicochemical environment and thereby directly influence the nucleosome and chromatin structures. Alternatively, histone PTMs can recruit effectors (or "readers") to signal the downstream events. A "histone code" hypothesis has been proposed in which the combinatory actions of different histone PTMs orchestrate the epigenetic landscape of cells, modulating the activity of the underlying DNA and maintaining the genome stability between generations. Accumulating evidence also suggests that malfunctions of histone PTMs are associated with the pathogenesis of human diseases, such as cancer. It is therefore important to fully decipher the histone code, namely, to dissect the regulatory mechanisms and biological functions of histone PTMs.Owing to the advances in state-of-the-art mass spectrometry, dozens of novel histone modifications have been archived during the past decade. However, most of these newly identified histone PTMs remain poorly explored. To unravel the roles played by these PTMs in histone code, key questions that have driven our study are (i) how to detect the novel histone PTMs; (ii) how to identify the enzymes that catalyze the addition (writers) and removal (erasers) of the histone PTMs along with the regulating mechanisms; (iii) what is the biological significance of the histone PTMs and how do they function, by affecting the nucleosome and chromatin dynamics or by recruiting readers; and (iv) how to develop chemical probes to interrogate the histone PTMs or even serve as potential leads for the drug discovery campaigns to treat diseases caused by abnormalities in the regulation of histone PTMs.This Account focuses on our efforts in developing and applying chemical tools and methods to answer the above questions. Specifically, we review the detection of negatively charged histone acylations by developing and applying chemical reporters; preparing homogeneous nucleosomes carrying negatively charged acylations by protein chemistry approaches and the in vitro biophysical analyses of the effects of the acylations on nucleosome structures; investigating the negatively charged acylations' influence on chromatin dynamics in vivo using yeast genetic approaches; identifying and characterizing protein-protein interactions (PPIs) mediated by histone PTMs in different biological contexts (i.e., to identify the readers and erasers) by establishing a chemical proteomics platform that is enabled by photo-cross-linking chemistry and quantitative proteomics strategies; and manipulating PTM-mediated PPIs by the structure-guided design of inhibitors. We also discuss possible future directions in our journey to fully decipher the histone code.

Analysis of nucleoid-associated protein-binding regions reveals DNA structural features influencing genome organization in Mycobacteriumtuberculosis.

Nucleoid-associated proteins (NAPs) maintain bacterial nucleoid configuration through their architectural properties of DNA bending, wrapping, and bridging. However, the contribution of DNA structural alterations to DNA-NAP recognition at the genomic scale remains unresolved. Present work dissects the DNA sequence, shape and altered structural preferences at a genomic scale for six NAPs in Mycobacteriumtuberculosis. Results suggest narrower minor groove width (MGW) and higher DNA rigidity are marked for the binding sites of EspR and Lsr2, while mIHF, MtHU and NapM haveheterogeneous DNA structural predilections. In contrast, WhiB4-DNA-binding sites were characterized by wider MGW, highly deformable and less curved DNA. This work provides systematic insight into NAP-mediated genome organization as a function of DNA structural features.

Mechanisms of SSBP1 variants in mitochondrial disease: Molecular dynamics simulations reveal stable tetramers with altered DNA binding surfaces

Several mutations in the gene for the mitochondrial single stranded DNA binding protein (SSBP1) have recently been implicated in human disease, but initial reports are insufficient to explain the molecular mechanism of disease, including the possible role of SSBP1 heterotetramers in heterozygous patients. Here we employed molecular simulations to model the dynamics of wild type and 31 variant SSBP1 tetramer systems, including 7 variant homotetramer and 24 representative heterotetramer systems. Our simulations indicate that all variants are stable and most have stronger intermonomer interactions, reduced solvent accessible surface areas, and a net loss of positive surface charge. We then used structural alignments and phosphate binding simulations to predict DNA binding surfaces on SSBP1. Our models suggest that nearly the entire surface of SSBP1, excluding flexible loops and protruding helices, is available for DNA binding, and we observed several potential DNA binding hotspots. Changes to the protein surface in variant SSBP1 tetramers potentially alter anchor points or wrapping paths, rather than abolishing binding altogether. Overall, our findings disqualify tetramer destabilization or gross disruption of DNA binding as mechanisms of disease. Instead, they are consistent with subtle changes to DNA binding, wrapping, or release that cause rare but consequential failures of mtDNA maintenance, which, in turn, are consistent with the late onset of disease in most of the reported SSBP1 cases.

Effect of DNA Flexibility on Complex Formation of a Cationic Nanoparticle with Double-Stranded DNA.

We present extensive molecular dynamics simulations of a cationic nanoparticle and a double-stranded DNA molecule to discuss the effect of DNA flexibility on the complex formation of a cationic nanoparticle with double-stranded DNA. Martini coarse-grained models were employed to describe double-stranded DNA molecules with two different flexibilities and cationic nanoparticles with three different electric charges. As the electric charge of a cationic nanoparticle increases, the degree of DNA bending increases, eventually leading to the wrapping of DNA around the nanoparticle at high electric charges. However, a small increase in the persistence length of DNA by 10 nm requires a cationic nanoparticle with a markedly increased electric charge to bend and wrap DNA around. Thus, a more flexible DNA molecule bends and wraps around a cationic nanoparticle with an intermediate electric charge, whereas a less flexible DNA molecule binds to a nanoparticle with the same electric charge without notable bending. This work provides solid evidence that a small difference in DNA flexibility (as small as 10 nm in persistence length) has a substantial influence on the complex formation of DNA with proteins from a biological perspective and suggests that the variation of sequence-dependent DNA flexibility can be utilized in DNA nanotechnology as a new tool to manipulate the structure of DNA molecules mediated by nanoparticle binding.

Transcription factor NF-κB unravels nucleosomes

NF-κB is a transcription factor responsible for activating hundreds of genes in mammalian organisms. To accomplish its function, NF-κB must interact with DNA occupied by nucleosomes, but how this interaction occurs is unclear. Here we used Atomic Force Microscopy to characterize complexes of NF-κB with nucleosomes assembled on different DNA templates. The assembly of NF-κB-nucleosome complexes leads to a substantial decrease of DNA wrapping efficiency from 149 ± 2 bp (SEM) for the control nucleosome sample to 135 ± 3 bp for complexes of nucleosomes with NF-κB. Mapping of the nucleosomes did not reveal displacement of under-wrapped nucleosomes from their original position, suggesting that unravelling involves dissociation of one or both flanks of the nucleosomes. Binding of NF-κB to the core was identified by nucleosome core volume measurements. We discovered two binding modes of NF-κB associated with nucleosome unravelling - NF-κB bound to the nucleosome core and to the DNA flanks. From these findings we propose two models explaining the interaction of NF-κB with the nucleosome complex. The partial unravelling of nucleosomes by NF-κB makes the DNA segment at the edge of the nucleosome core accessible, facilitating the transcription process. We speculate that NF-κB can function as a pioneer factor, enhancing its ability to facilitate rapid transcriptional response to cell stress.

Cryo-EM structure of SETD2/Set2 methyltransferase bound to a nucleosome containing oncohistone mutations

Substitution of lysine 36 with methionine in histone H3.3 (H3.3K36M) is an oncogenic mutation that inhibits SETD2-mediated histone H3K36 tri-methylation in tumors. To investigate how the oncohistone mutation affects the function of SETD2 at the nucleosome level, we determined the cryo-EM structure of human SETD2 associated with an H3.3K36M nucleosome and cofactor S-adenosylmethionine (SAM), and revealed that SETD2 is attached to the N-terminal region of histone H3 and the nucleosome DNA at superhelix location 1, accompanied with the partial unwrapping of nucleosome DNA to expose the SETD2-binding site. These structural features were also observed in the previous cryo-EM structure of the fungal Set2–nucleosome complex. By contrast with the stable association of SETD2 with the H3.3K36M nucleosome, the EM densities of SETD2 could not be observed on the wild-type nucleosome surface, suggesting that the association of SETD2 with wild-type nucleosome might be transient. The linker histone H1, which stabilizes the wrapping of nucleosome DNA at the entry/exit sites, exhibits an inhibitory effect on the activities of SETD2 and displays inversely correlated genome distributions with that of the H3K36me3 marks. Cryo-EM analysis of yeast H3K36 methyltransferase Set2 complexed with nucleosomes further revealed evolutionarily conserved structural features for nucleosome recognition in eukaryotes, and provides insights into the mechanism of activity regulation. These findings have advanced our understanding of the structural basis for the tumorigenesis mechanism of the H3.3K36M mutation and highlight the effect of nucleosome conformation on the regulation of histone modification.

The Effects of Site Specific Histone Modifications on Nucleosomal DNA Accessibility

Chromatin accessibility is the ability of nucleosomal DNA to be physically accessed by cellular biomachinery. Accessibility of chromatin is controlled by a slew of post translational modifications to histone proteins, DNA, and chromatin-binding factors. This growing field of research has become increasingly important as chromatin accessibility has a large effect on transcription and gene regulation. Gene and cell cycle regulation must be understood to properly study their misregulation which commonly occurs in most, if not all, cancers. Thus, understanding the organization and regulation of the chromatin state by post translational modifications plays an important role in understanding the dysfunction of cancerous cells. The most basic unit of chromatin is the nucleosome core particle. The nucleosome core particle is made up of an octamer that has two copies each of the 4 histone proteins H2A, H2B, H3, and H4 with DNA wrapping around the octamer itself. It is on the N-terminal tails of these histone proteins that many post-translational modifications take place the regulate the chromatin state. In this study, the effect of histone lysine acetylation on the DNA-histone core interaction will be assessed by its thermal stability and accessibility of nucleosomal DNA to Pst1 Digestion. We propose to assay singly acetylated mononucleosomes at the following sites: H2A: K5; H2B: K5, K12, K15, and K20; H3: K9, K14, K18, K23, K27, K36, K37, K64, K56, K79, K115, and K122; H4: K5, K8, K12, and K16. We predict that, as previously shown with nu′-H3K18ac and nu′-H3K36ac acetylation at lysine sites will facilitate unwrapping of nucleosomal DNA.

Asymmetry in histone rotation in forced unwrapping and force quench rewrapping in a nucleosome.

Single molecule pulling experiments have shown that DNA in the nucleosomes unwraps in two stages from the histone protein core (HPC). The first stage, attributed to the rupture of the outer DNA turn, occurs between 3 and 5 pNs, and is reversible. The inner DNA turn ruptures irreversibly at forces between 9 and 15 pNs (or higher) in the second stage. Molecular simulations using the Self-Organized Polymer model capture the experimental findings. The unwrapping of the outer DNA turn is independent of the pulling direction. The rupture of the DNA inner turn depends on the pulling direction and involves overcoming substantial energetic (most likely electrostatic in origin) and kinetic barriers. They arise because the mechanical force has to generate sufficient torque to rotate the HPC by 180°. On the other hand, during the rewrapping process, HPC rotation is stochastic, with force playing no role. The assembly of the outer DNA wrap upon force quench nearly coincides with the unwrapping process, confirming the reversibility of the outer turn rupture. The asymmetry in HPC rotation during unwrapping and rewrapping explains the observed hysteresis in the stretch-release cycles in experiments. We propose experiments to test the prediction that HPC rotation produces kinetic barriers in the unwrapping process.

Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways.

Eukaryotes and many archaea package their DNA with histones. While the four eukaryotic histones wrap ~147 DNA base pairs into nucleosomes, archaeal histones form 'nucleosome-like' complexes that continuously wind between 60 and 500 base pairs of DNA ('archaeasomes'), suggested by crystal contacts and analysis of cellular chromatin. Solution structures of large archaeasomes (>90 DNA base pairs) have never been directly observed. Here, we utilize molecular dynamics simulations, analytical ultracentrifugation, and cryoEM to structurally characterize the solution state of archaeasomes on longer DNA. Simulations reveal dynamics of increased accessibility without disruption of DNA-binding or tetramerization interfaces. Mg2+ concentration influences compaction, and cryoEM densities illustrate that DNA is wrapped in consecutive substates arranged 90o out-of-plane with one another. Without ATP-dependent remodelers, archaea may leverage these inherent dynamics to balance chromatin packing and accessibility.

Rtt109 slows replication speed by histone N-terminal acetylation.

The wrapping of DNA around histone octamers challenges processes that use DNA as their template. In vitro, DNA replication through chromatin depends on histone modifiers, raising the possibility that cells modify histones to optimize fork progression. Rtt109 is an acetyl transferase that acetylates histone H3 before its DNA incorporation on the K56 and N-terminal residues. We previously reported that, in budding yeast, a wave of histone H3 K9 acetylation progresses ∼3–5 kb ahead of the replication fork. Whether this wave contributes to replication dynamics remained unknown. Here, we show that the replication fork velocity increases following deletion of RTT109, the gene encoding the enzyme required for the prereplication H3 acetylation wave. By using histone H3 mutants, we find that Rtt109-dependent N-terminal acetylation regulates fork velocity, whereas K56 acetylation contributes to replication dynamics only when N-terminal acetylation is compromised. We propose that acetylation of newly synthesized histones slows replication by promoting replacement of nucleosomes evicted by the incoming fork, thereby protecting genome integrity.

Molecular mechanism of the MORC4 ATPase activation

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.

Structural basis of nucleosome dynamics modulation by histone variants H2A.B andH2A.Z.2.2.

Nucleosomes are dynamic entities with wide-ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo-EM structures of nucleosome core particles containing human H2A.B (H2A.B-NCP) at atomic resolution, identifying large-scale structural rearrangements in the histone octamer in H2A.B-NCP. H2A.B-NCP compacts approximately 103bp of DNA wrapping around the core histones in approximately 1.2 left-handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B-specific residues, including a ROF ("regulating-octamer-folding") sequence of six consecutive residues, are responsible for loosening of H2A.B-NCPs. Unlike H2A.B-NCP, the H2A.Z.2.2-containing nucleosome (Z.2.2-NCP) adopts a less-extended structure and compacts around 125bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2-specific ROF in both H2A.Z.2.2-NCP opening and SWR1-dependent histone replacement. Taken together, these first high-resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.

An analytical method to connect open curves for modeling protein-bound DNA minicircles

We introduce an analytical method to generate the pathway of a closed protein-bound DNA minicircle. We develop an analytical equation to connect two open curves smoothly and use the derived expressions to join the ends of two helical pathways and form models of nucleosome-decorated DNA minicircles. We find that the simplest smooth connector which satisfies the boundary conditions at the end points and the length requirement for such connections to be a quartic function on the xy-plane and linear along the z-direction. This is a general method which can be used to connect any two open curves with well defined mathematical definitions as well as pairs of discrete systems found experimentally. We used this method to describe the configurations of torsionally relaxed, 360-base pair DNA rings with two evenly-spaced, ideal nucleosomes. We considered superhelical nucleosomal pathways with different levels of DNA wrapping and allowed for different inter-nucleosome orientations. We completed the DNA circles with the smooth connectors and studied the associated bending and electrostatic energies for different configurations in the absence and presence of salt. The predicted stable states bear close resemblance to reconstituted minicircles observed under low and high salt conditions.

The pivot point arginines identified in the \u03b2-pinwheel structure of C-terminal domain from Salmonella Typhi DNA Gyrase A subunit

The essentiality of DNA Gyrase in basic cellular processes in bacterial pathogens makes it an ideal drug target. Though the Gyrase has a conserved mechanism of action, the complete DNA wrapping and binding process is still unknown. In this study, we have identified six arginine residues R556, R612, R667, R716, R766, and R817 in the DNA GyraseA – C-terminal domain from Salmonella enterica serovar Typhi (StGyrA-CTD) to be essential for DNA wrapping and sliding by a sequence and structure analysis. Through site-directed mutagenesis and EMSA studies, we observed that the substitution of R667 (blade 3) and R716 (blade 4) in StGyrA-CTD led to loss of DNA binding. Whereas, upon mutation of residue R612 (blade2), R766 (blade5) and R817 (blade6) along with supporting residue R712 (blade 4) a decrease in binding affinity was seen. Our results indicate that R667 and R716 act as a pivot point in DNA wrapping and sliding during gyrase catalytic activity. In this study, we propose that the DNA wrapping mechanism commences with DNA binding at blade3 and blade4 followed by other blades to facilitate the DNA sliding during supercoiling activity. This study provides a better understanding of the DNA binding and wrapping mechanism of GyrA-CTD in DNA Gyrase.

Histone H1 Remains Bound to the Nucleosome Dyad During TF Binding within a Nucleosome

H1 is a lysine rich histone that binds nucleosomes and alters nucleosome/chromatin dynamics. Chromatin dynamics, such as chromatin compaction and nucleosome unwrapping, are integral for regulation of fundamental cellular processes including transcription, DNA replication, and DNA repair. Nucleosomal DNA in the entry/exit region spontaneously dissociates from the histone octamer allowing transcription factors (TFs) to bind to DNA in the region when the nucleosome is in an unwrapped state. Instead of blocking wrapping, H1 shifts this equilibrium towards a wrapped state which represses TF binding without fully occluding it. Details of this TF binding mechanism remain unknown, including the structural dynamics of H1 domains during TF binding and which domains affect wrapping and TF binding. Using in vitro fluorescence‐based assays, we have determined the structural dynamics of H1 during TF binding to a nucleosome. H1 binds nucleosomes via electrostatic interactions with DNA and is composed of 3 domains: The winged helix domain that binds the nucleosome dyad and linker DNA, and the intrinsically disordered C terminal and N terminal domains that bind linker DNA. Previous work implied that H1 remains bound to a nucleosome during TF binding, but the details of H1 domain locations were unexplored. We have attached fluorophores and fluorophore quenchers to all 3 H1 domains and either the DNA or histone octamer to measure FRET and fluorophore quenching. This allows us to monitor the movement of the H1 domains relative to the DNA and histone octamer during TF binding. Our results indicate that H1 remains bound to the nucleosome dyad during a TF binding event, and the H1 C terminal domain dissociates from the linker DNA to remain at the dyad. These data further understanding of the mechanistic details of H1 suppression of TF binding to nucleosomes. Future experiments will focus on which H1 domains affect DNA wrapping and TF binding to a nucleosome to further understanding of how H1 regulates TF binding and the implications for transcription.Support or Funding InformationNIH R01‐GM121966NIH T32‐GM086252OSU University Fellowship