Nucleosomes, the basic unit of chromatin, package and regulate expression of eukaryotic genomes. Although the structure of the intact nucleosome has been studied, little is known about structures of its partially unwrapped, transient intermediates. In this study, we present 9 cryo EM structures of distinct conformations of nucleosome and subnucleosome particles. Our structures show that initial DNA breathing induces conformational changes in the histone octamer, particularly in histone H3, that propagate through the nucleosome and prevent symmetrical DNA opening. Rearrangements in the H2A–H2B dimer strengthen interaction with the unwrapping DNA and promote nucleosome stability. In agreement, cross-linked H2A–H2B that can not accommodate to the unwrapping of the DNA is not stably maintained in the nucleosome. H2A–H2B release and DNA unwrapping occur simultaneously indicating that DNA is essential in stabilizing the dimer in the nucleosome. Our structures reveal intrinsic nucleosomal plasticity that is required for nucleosome stability and might be exploited by extrinsic protein factors.

Methanogenic archaea possess unusual seryl-tRNA synthetase (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. The two types of SerRSs show only minimal sequence similarity, primarily within class II conserved motifs 1, 2 and 3. Here, we report a 2.5 Å resolution crystal structure of the atypical methanogenic Methanosarcina barkeri SerRS and its complexes with ATP, serine and the nonhydrolysable seryl-adenylate analogue 5 0 -O-(N-serylsulfamoyl)adenosine. The structures reveal two idiosyncratic features of methanogenic SerRSs: a novel N-terminal tRNA-binding domain and an active site zinc ion. The tetra-coordinated Zn 2 þ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461) and binds the amino group of the serine substrate. The absolute requirement of the metal ion for enzymatic activity was confirmed by mutational analysis of the direct zinc ion ligands. This zinc-dependent serine recognition mechanism differs fundamentally from the one employed by the bacterial-type SerRSs. Consequently, SerRS represents the only known aminoacyl-tRNA synthetase system that evolved two distinct mechanisms for the recognition of the same amino-acid substrate.

Nucleosomes, the basic unit of chromatin, package and regulate expression of eukaryotic genomes. Nucleosomes are highly dynamic and are remodeled with the help of ATP-dependent remodeling factors. Yet, the mechanism of DNA translocation around the histone octamer is poorly understood. In this study, we present several nucleosome structures showing histone proteins and DNA in different organizational states. We observe that the histone octamer undergoes conformational changes that distort the overall nucleosome structure. As such, rearrangements in the histone core α-helices and DNA induce strain that distorts and moves DNA at SHL 2. Distortion of the nucleosome structure detaches histone α-helices from the DNA, leading to their rearrangement and DNA translocation. Biochemical assays show that cross-linked histone octamers are immobilized on DNA, indicating that structural changes in the octamer move DNA. This intrinsic plasticity of the nucleosome is exploited by chromatin remodelers and might be used by other chromatin machineries.

Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved enzymes catalyzing the formation of aminoacyl-tRNAs, that are used as substrates for ribosomal protein biosynthesis. In addition to full length aaRS genes, genomes of many organisms are sprinkled with truncated genes encoding single-domain aaRSlike proteins, which often have relinquished their canonical role in genetic code translation. We have identified the genes for putative seryl-tRNA synthetase homologs widespread in bacterial genomes and characterized three of them biochemically and structurally. The proteins encoded are homologous to the catalytic domain of highly diverged, atypical seryl-tRNA synthetases (aSerRSs) found only in methanogenic archaea and are deprived of the tRNA-binding domain. Remarkably, in comparison to SerRSs, aSerRS homologs display different and relaxed amino acid specificity. aSerRS homologs lack canonical tRNA aminoacylating activity and instead transfer activated amino acid to phosphopantetheine prosthetic group of putative carrier proteins, whose genes were identified in the genomic surroundings of aSerRS homologs. Detailed kinetic analysis confirmed that aSerRS homologs aminoacylate these carrier proteins efficiently and specifically. Accordingly, aSerRS homologs were renamed amino acid:[carrier protein] ligases (AMP forming). The enzymatic activity of aSerRS homologs is reminiscent of adenylation domains in nonribosomal peptide synthesis, and thus they represent an intriguing link between programmable ribosomal protein biosynthesis and template-independent nonribosomal peptide synthesis.

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous assemblies composed of multiple copies of ∼30 different proteins called nucleoporins. To unravel the basic scaffold underlying the NPC, we have characterized the speciesspecific scaffold nucleoporin Nup37 and ELY5/ELYS. Both proteins integrate directly via Nup120/160 into the universally conserved heptameric Y-complex, the critical unit for the assembly and functionality of the NPC. We present the crystal structure of Schizosaccharomyces pombe Nup37 in complex with Nup120, a 174-kDa subassembly that forms one of the two short arms of the Y-complex. Nup37 binds near the bend of the L-shaped Nup120 protein, potentially stabilizing the relative orientation of its two domains. By means of reconstitution assays, we pinpoint residues crucial for this interaction. In vivo and in vitro results show that ELY5 binds near an interface of the Nup120-Nup37 complex. Complementary biochemical and cell biological data refine and consolidate the interactions of Nup120 within the current Y-model. Finally, we propose an orientation of the Y-complex relative to the pore membrane, consistent with the lattice model. macromolecular assemblies | structural biology

Summary: DNA strand breaks recruit PARP1 and its paralogue PARP2 to modify histones and many other substrates with mono- and poly(ADP-ribose) (PAR) 1 – 5 . In DNA damage response, the PAR post-translational modification occurs predominantly on serine amino acids 6 – 8 , which requires HPF1, an accessory factor that switches the amino-acid specificity of PARP1/2 from aspartate/glutamate to serine residues 9 , 10 . Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and serves as an anchor to recruit a variety of downstream signaling and repair factors to the sites of DNA breaks 2 , 11 . To understand the molecular mechanism of DNA break recognition by PARP enzymes in the context of chromatin, we determined cryo-EM structure of PARP2/HPF1 bound to a nucleosome. The structure shows that PARP2/HPF1 bridges two nucleosomes, with the broken DNA aligned in a ligation-competent position, revealing the initial step in double-strand DNA break repair. The bridging induces structural changes in PARP2 that signal DNA break recognition to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD + and substrate, which may enable PARP enzymes to be processive while bound to chromatin. The mechanisms of PARP activation and catalytic cycle we describe can explain resistance mechanisms to PARP inhibitors, and will aid development of better inhibitors for cancer treatments 12 – 16 .

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification deposited by the Set2 methyltransferases. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly understood. Set2 enzymes show spurious activity on histones and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Our structure shows that Set2 makes extensive interactions with the H3 αN, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies.

Centromeres are defined epigenetically by nucleosomes containing the histone H3 variant CENP‐A, upon which the constitutive centromere‐associated network of proteins (CCAN) is built. CENP‐C is considered to be a central organizer of the CCAN. We provide new molecular insights into the structure of human CENP‐A nucleosomes, in isolation and in complex with the CENP‐C central region (CENP‐CCR), the main CENP‐A binding module of human CENP‐C. We establish that the short αN helix of CENP‐A promotes DNA flexibility at the nucleosome ends, independently of the sequence it wraps. Furthermore, we show that, in vitro, two regions of human CENP‐C (CENP‐CCR and CENP‐Cmotif) both bind exclusively to the CENP‐A nucleosome. We find CENP‐CCR to bind with high affinity due to an extended hydrophobic area made up of CENP‐AV532 and CENP‐AV533. Importantly, we identify two key conformational changes within the CENP‐A nucleosome upon CENP‐C binding. First, the loose DNA wrapping of CENP‐A nucleosomes is further exacerbated, through destabilization of the H2A C‐terminal tail. Second, CENP‐CCR rigidifies the N‐terminal tail of H4 in the conformation favoring H4K20 monomethylation, essential for a functional centromere.