CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail

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-C-CR), the main CENP-A binding module of human CENP-C. We establish that the short alpha N 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-C-CR and CENP-C-motif) both bind exclusively to the CENP-A nucleosome. We find CENP-C-CR to bind with high affinity due to an extended hydrophobic area made up of CENP-A(V)(532) and CENP-A(V)(533). 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-C-CR rigidifies the N-terminal tail of H4 in the conformation favoring H4(K20) monomethylation, essential for a functional centromere

Molecular Evolution of Aminoacyl tRNA Synthetase Proteins in the Early History of Life

Aminoacyl-tRNA synthetases (aaRS) consist of several families of functionally conserved proteins essential for translation and protein synthesis. Like nearly all components of the translation machinery, most aaRS families are universally distributed across cellular life, being inherited from the time of the Last Universal Common Ancestor (LUCA). However, unlike the rest of the translation machinery, aaRS have undergone numerous ancient horizontal gene transfers, with several independent events detected between domains, and some possibly involving lineages diverging before the time of LUCA. These transfers reveal the complexity of molecular evolution at this early time, and the chimeric nature of genomes within cells that gave rise to the major domains. Additionally, given the role of these protein families in defining the amino acids used for protein synthesis, sequence reconstruction of their pre-LUCA ancestors can reveal the evolutionary processes at work in the origin of the genetic code. In particular, sequence reconstructions of the paralog ancestors of isoleucyl- and valyl- RS provide strong empirical evidence that at least for this divergence, the genetic code did not co-evolve with the aaRSs; rather, both amino acids were already part of the genetic code before their cognate aaRSs diverged from their common ancestor. The implications of this observation for the early evolution of RNA-directed protein biosynthesis are discussed.National Science Foundation (U.S.) (Grant DEB 0830024)National Science Foundation (U.S.) (Grant DEB 0936234)United States. National Aeronautics and Space Administration (NASA Postdoctoral Fellowship

Atomic Resolution Cryo-EM Structure Of A Nativelike CENP-A Nucleosome Aided By An Antibody Fragment

Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences

Ancient horizontal gene transfer and the last common ancestors

Background The genomic history of prokaryotic organismal lineages is marked by extensive horizontal gene transfer (HGT) between groups of organisms at all taxonomic levels. These HGT events have played an essential role in the origin and distribution of biological innovations. Analyses of ancient gene families show that HGT existed in the distant past, even at the time of the organismal last universal common ancestor (LUCA). Most gene transfers originated in lineages that have since gone extinct. Therefore, one cannot assume that the last common ancestors of each gene were all present in the same cell representing the cellular ancestor of all extant life. Results Organisms existing as part of a diverse ecosystem at the time of LUCA likely shared genetic material between lineages. If these other lineages persisted for some time, HGT with the descendants of LUCA could have continued into the bacterial and archaeal lineages. Phylogenetic analyses of aminoacyl-tRNA synthetase protein families support the hypothesis that the molecular common ancestors of the most ancient gene families did not all coincide in space and time. This is most apparent in the evolutionary histories of seryl-tRNA synthetase and threonyl-tRNA synthetase protein families, each containing highly divergent “rare” forms, as well as the sparse phylogenetic distributions of pyrrolysyl-tRNA synthetase, and the bacterial heterodimeric form of glycyl-tRNA synthetase. These topologies and phyletic distributions are consistent with horizontal transfers from ancient, likely extinct branches of the tree of life. Conclusions Of all the organisms that may have existed at the time of LUCA, by definition only one lineage is survived by known progeny; however, this lineage retains a genomic record of heterogeneous genetic origins. The evolutionary histories of aminoacyl-tRNA synthetases (aaRS) are especially informative in detecting this signal, as they perform primordial biological functions, have undergone several ancient HGT events, and contain many sites with low substitution rates allowing deep phylogenetic reconstruction. We conclude that some aaRS families contain groups that diverge before LUCA. We propose that these ancient gene variants be described by the term “hypnologs”, reflecting their ancient, reticulate origin from a time in life history that has been all but erased”.National Science Foundation (U.S.) (Grant DEB 0830024)Exobiology Program (U.S.) (Grant NNX10AR85G)United States. National Aeronautics and Space Administration (Postdoctoral Program

Histone octamer rearranges to adapt to DNA unwrapping

Nucleosomes, the basic units of chromatin, package and regulate expression of eukaryotic genomes. Although the structure of the intact nucleosome is well characterized, little is known about structures of partially unwrapped, transient intermediates. In this study, we present nine cryo-EM structures of distinct conformations of nucleosome and subnucleo-some particles. These 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 with this, cross-linked H2A-H2B that cannot accommodate 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

Bridging of DNA breaks activates PARP2–HPF1 to modify chromatin

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1,2,3,4,5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6,7,8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2–HPF1 bound to a nucleosome. This showed that PARP2–HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break 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 act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12,13,14,15,16

Homologs of aminoacyl-tRNA synthetases acylate carrier proteins and provide a link between ribosomal and nonribosomal peptide synthesis

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 aaRS-like 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