Virus-encoded histone doublets are essential and form nucleosome-like structures.

The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of "minimalist" histones in archaea and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones are essential for viral infectivity, localize to cytoplasmic viral factories after virus infection, and ultimately are found in the mature virions. Cryogenic electron microscopy (cryo-EM) structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of "organisms" that require nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.

BEES: Bayesian Ensemble Estimation from SAS.

Many biomolecular complexes exist in a flexible ensemble of states in solution that is necessary to perform their biological function. Small-angle scattering (SAS) measurements are a popular method for characterizing these flexible molecules because of their relative ease of use and their ability to simultaneously probe the full ensemble of states. However, SAS data is typically low dimensional and difficult to interpret without the assistance of additional structural models. In theory, experimental SAS curves can be reconstituted from a linear combination of theoretical models, although this procedure carries a significant risk of overfitting the inherently low-dimensional SAS data. Previously, we developed a Bayesian-based method for fitting ensembles of model structures to experimental SAS data that rigorously avoids overfitting. However, we have found that these methods can be difficult to incorporate into typical SAS modeling workflows, especially for users that are not experts in computational modeling. To this end, we present the Bayesian Ensemble Estimation from SAS (BEES) program. Two forks of BEES are available, the primary one existing as a module for the SASSIE web server and a developmental version that is a stand-alone Python program. BEES allows users to exhaustively sample ensemble models constructed from a library of theoretical states and to interactively analyze and compare each model's performance. The fitting routine also allows for secondary data sets to be supplied, thereby simultaneously fitting models to both SAS data as well as orthogonal information. The flexible ensemble of K63-linked ubiquitin trimers is presented as an example of BEES' capabilities.

Effects of MacroH2A and H2A.Z on Nucleosome Dynamics as Elucidated by Molecular Dynamics Simulations.

Eukaryotes tune the transcriptional activity of their genome by altering the nucleosome core particle through multiple chemical processes. In particular, replacement of the canonical H2A histone with the variants macroH2A and H2A.Z has been shown to affect DNA accessibility and nucleosome stability; however, the processes by which this occurs remain poorly understood. In this study, we elucidate the molecular mechanisms of these variants with an extensive molecular dynamics study of the canonical nucleosome along with three variant-containing structures: H2A.Z, macroH2A, and an H2A mutant with macroH2A-like L1 loops. Simulation results show that variant L1 loops play a pivotal role in stabilizing DNA binding to the octamer through direct interactions, core structural rearrangements, and altered allosteric networks in the nucleosome. All variants influence dynamics; however, macroH2A-like systems have the largest effect on energetics. In addition, we provide a comprehensive analysis of allosteric networks in the nucleosome and demonstrate that variants take advantage of stronger interactions between L1 loops to propagate dynamics throughout the complex. Furthermore, we show that posttranslational modifications are enriched at key locations in these networks. Taken together, these results provide, to our knowledge, new insights into the relationship between the structure, dynamics, and function of the nucleosome core particle and chromatin fibers, and how they are influenced by chromatin remodeling factors.

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging.

PARP1 contributes to genome architecture and DNA damage repair through its dynamic association with chromatin. PARP1 and PARP2 (PARP1/2) recognize damaged DNA and recruit the DNA repair machinery. Using single-molecule microscopy in live cells, we monitored the movement of PARP1/2 on undamaged and damaged chromatin. We identify two classes of freely diffusing PARP1/2 and two classes of bound PARP1/2. The majority (>60%) of PARP1/2 diffuse freely in both undamaged and damaged nuclei and in the presence of inhibitors of PARP1/2 used for cancer therapy (PARPi). Laser-induced DNA damage results in a small fraction of slowly diffusing PARP1 and PARP2 to become transiently bound. Treatment of cells with PARPi in the presence of DNA damage causes subtle changes in the dynamics of bound PARP1/2, but not the high levels of PARP1/2 trapping seen previously. Our results imply that next-generation PARPi could specifically target the small fraction of DNA-bound PARP1/2.

Automated modeling of protein accumulation at DNA damage sites using qFADD.py.

Eukaryotic cells are constantly subject to DNA damage, often with detrimental consequences for the health of the organism. Cells mitigate this DNA damage through a variety of repair pathways involving a diverse and large number of different proteins. To better understand the cellular response to DNA damage, one needs accurate measurements of the accumulation, retention, and dissipation timescales of these repair proteins. Here, we describe an automated implementation of the "quantitation of fluorescence accumulation after DNA damage" method that greatly enhances the analysis and quantitation of the widely used technique known as laser microirradiation, which is used to study the recruitment of DNA repair proteins to sites of DNA damage. This open-source implementation ("qFADD.py") is available as a stand-alone software package that can be run on laptops or computer clusters. Our implementation includes corrections for nuclear drift, an automated grid search for the model of a best fit, and the ability to model both horizontal striping and speckle experiments. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As a practical example, we present and discuss the recruitment dynamics of the early responder PARP1 to DNA damage sites.

Archaea: The Final Frontier of Chromatin.

The three domains of life employ various strategies to organize their genomes. Archaea utilize features similar to those found in both eukaryotic and bacterial chromatin to organize their DNA. In this review, we discuss the current state of research regarding the structure-function relationships of several archaeal chromatin proteins (histones, Alba, Cren7, and Sul7d). We address individual structures as well as inferred models for higher-order chromatin formation. Each protein introduces a unique phenotype to chromatin organization, and these structures are put into the context of in vivo and in vitro data. We close by discussing the present gaps in knowledge that are preventing further studies of the organization of archaeal chromatin, on both the organismal and domain level.

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.

All animals, plants and fungi belong to a group of living organisms called eukaryotes. The two other groups are bacteria and archaea, which include unicellular, microscopic organisms. All three groups have genes, which are typically stored on long strands of DNA. Eukaryotes have so much DNA that they use proteins called histones to help package and organize it inside each cell. Archaea also have simplified histones that help store their DNA, and studying these proteins could reveal how eukaryotic histones first evolved. In eukaryotes, groups of eight histones form a short cylinder that organizes a small section of DNA into a structure called a nucleosome. Each cell needs hundreds of thousands of nucleosomes to arrange its DNA. Eukaryotic cells also contain other proteins that release pieces of DNA from histones so that their genetic information can be used. The histones in Archaea don't form discrete nucleosomes, instead, they coil DNA into 'slinky-like' shapes. It's still unclear how DNA packing in archaea works and how it differs from eukaryotes. Bowerman, Wereszczynski and Luger used computer simulations, biochemistry and cryo-electron microscopy to study the histones from archaea. The archaeal 'slinky-like' histone structures are more flexible than nucleosomes, and can open and close like clamshells. This flexibility allows the information in the genomes of Archaea to be easily accessed, so, unlike in eukaryotes, archaeal cells may not need other proteins to release the DNA from the histones. The ability to package DNA allows cells to contain many more genes, so evolving histones was a vital step in the evolution of eukaryotic life, including the appearance of animals. Archaeal histones may reflect early versions of histones in eukaryotes, and can be used to understand how DNA packing has evolved. Furthermore, a greater understanding of Archaea may help better explain their role in health and global ecosystems, and allow their use in industrial applications.

Analytical Ultracentrifugation (AUC): An Overview of the Application of Fluorescence and Absorbance AUC to the Study of Biological Macromolecules.

The biochemical and biophysical investigation of proteins, nucleic acids, and the assemblies that they form yields essential information to understand complex systems. Analytical ultracentrifugation (AUC) represents a broadly applicable and information-rich method for investigating macromolecular characteristics such as size, shape, stoichiometry, and binding properties, all in the true solution-state environment that is lacking in most orthogonal methods. Despite this, AUC remains underutilized relative to its capabilities and potential in the fields of biochemistry and molecular biology. Although there has been a rapid development of computing power and AUC analysis tools in this millennium, fewer advancements have occurred in development of new applications of the technique, leaving these powerful instruments underappreciated and underused in many research institutes. With AUC previously limited to absorbance and Rayleigh interference optics, the addition of fluorescence detection systems has greatly enhanced the applicability of AUC to macromolecular systems that are traditionally difficult to characterize. This overview provides a resource for novices, highlighting the potential of AUC and encouraging its use in their research, as well as for current users, who may benefit from our experience. We discuss the strengths of fluorescence-detected AUC and demonstrate the power of even simple AUC experiments to answer practical and fundamental questions about biophysical properties of macromolecular assemblies. We address the development and utility of AUC, explore experimental design considerations, present case studies investigating properties of biological macromolecules that are of common interest to researchers, and review popular analysis approaches. © 2020 The Authors.

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1.

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

Unique Dynamics in Asymmetric macroH2A-H2A Hybrid Nucleosomes Result in Increased Complex Stability.

The fundamental unit of eukaryotic chromatin is the nucleosome core particle, a protein/DNA complex that binds ∼147 base pairs of DNA to a histone octamer. These histones-H3, H4, H2A, H2B-form the nucleosome core through a stacked interaction in which two H2A-H2B dimers flank the (H3-H4)2 tetramer. In vivo, genetic accessibility can be modulated by the substitution of canonical histones with variant proteins that contain the same structural motif but a different amino acid sequence, such as the transcriptional repression-associated macroH2A variant. Previously, Chakravarthy and Luger published a crystal study that showed that H2A substitution is not necessarily required of both H2A moieties, but that in vitro recombination of nucleosomes in the presence of both macroH2A and H2A histone folds results in a hybrid macroH2A-H2A nucleosome with one dimer of each type. Here, we present molecular dynamics simulations of this hybrid construct and compare the results to our previous study on homogeneous H2A- and macroH2A-containing nucleosomes. We find that the hybrid contains a unique set of dynamics that stabilize the interactions between protein constituents and create an altogether more stable nucleosome, both in terms of protein-DNA and protein-protein binding. While dimer-tetramer interactions are asymmetric, as the difference in moieties would suggest, we observe that it is the canonical dimer that is pulled further into the nucleosome core, resulting in more secure dimer-tetramer bonds and a more stable histone core, and we also find significantly more interaction between the dimer subunits. Together, these models provide evidence for hybrid H2A-macroH2A nucleosome formation being not only possible but actually energetically more favorable than a homogeneous construct, with dynamics that are unique from their homogeneous H2A or macroH2A nucleosome counterparts. These effects of hybrid substitution likely propagate into higher-order chromatin structures to hinder transcriptional activity.

Quantitating repair protein accumulation at DNA lesions: Past, present, and future.

All organisms must protect their genome from constantly occurring DNA damage. To this end, cells have evolved complex pathways for repairing sites of DNA lesions, and multiple in vitro and in vivo techniques have been developed to study these processes. In this review, we discuss the commonly used laser microirradiation method for monitoring the accumulation of repair proteins at DNA damage sites in cells, and we outline several strategies for deriving kinetic models from such experimental data. We discuss an example of how in vitro measurements and in vivo microirradation experiments complement each other to provide insight into the mechanism of PARP1 recruitment to DNA lesions. We also discuss a strategy to combine data obtained for the recruitment of many different proteins in a move toward fully quantitating the spatiotemporal relationships between various damage responses, and we outline potential venues for future development in the field.

The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome.

Histone tails harbor a plethora of post-translational modifications that direct the function of chromatin regulators, which recognize them through effector domains. Effector domain/histone interactions have been broadly studied, but largely using peptide fragments of histone tails. Here, we extend these studies into the nucleosome context and find that the conformation adopted by the histone H3 tails is inhibitory to BPTF PHD finger binding. Using NMR spectroscopy and MD simulations, we show that the H3 tails interact robustly but dynamically with nucleosomal DNA, substantially reducing PHD finger association. Altering the electrostatics of the H3 tail via modification or mutation increases accessibility to the PHD finger, indicating that PTM crosstalk can regulate effector domain binding by altering nucleosome conformation. Together, our results demonstrate that the nucleosome context has a dramatic impact on signaling events at the histone tails, and highlights the importance of studying histone binding in the context of the nucleosome.

Determining Atomistic SAXS Models of Tri-Ubiquitin Chains from Bayesian Analysis of Accelerated Molecular Dynamics Simulations.

Small-angle X-ray scattering (SAXS) has become an increasingly popular technique for characterizing the solution ensemble of flexible biomolecules. However, data resulting from SAXS is typically low-dimensional and is therefore difficult to interpret without additional structural knowledge. In theory, molecular dynamics (MD) trajectories can provide this information, but conventional simulations rarely sample the complete ensemble. Here, we demonstrate that accelerated MD simulations can be used to produce higher quality models in shorter time scales than standard simulations, and we present an iterative Bayesian Monte Carlo method that is able to identify multistate ensembles without overfitting. This methodology is applied to several ubiquitin trimers to demonstrate the effect of linkage type on the solution states of the signaling protein. We observe that the linkage site directly affects the solution flexibility of the trimer and theorize that this difference in plasticity contributes to their disparate roles in vivo.