Franziska Bleichert

Werner protein protects nonproliferating cells from oxidative DNA damage

ABSTRACT Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of γH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

The heterohexameric minichromosome maintenance (MCM2–7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2–7 particle is thought to open to facilitate loading onto DNA. The conformational state accessed during ring opening, the interplay between ATP binding and MCM2–7 architecture, and the use of these events in the regulation of DNA unwinding are poorly understood. To address these issues in isolation from the regulatory complexity of existing eukaryotic model systems, we investigated the structure/function relationships of a naturally minimized MCM2–7 complex from the microsporidian parasite Encephalitozoon cuniculi. Electron microscopy and small-angle X-ray scattering studies show that, in the absence of ATP, MCM2–7 spontaneously adopts a left-handed, open-ring structure. Nucleotide binding does not promote ring closure but does cause the particle to constrict in a two-step process that correlates with the filling of high- and low-affinity ATPase sites. Our findings support the idea that an open ring forms the default conformational state of the isolated MCM2–7 complex, and they provide a structural framework for understanding the multiphasic ATPase kinetics observed in different MCM2–7 systems.

A Dimeric Structure for Archaeal Box C/D Small Ribonucleoproteins

Seeing Double A particular set of ubiquitous small (nucleolar) ribonucleoproteins are important for optimal ribosome function and protein synthesis. Bleichert et al. (p. 1384) used electron microscopy and single-particle analysis to investigate the structure of an archaeal version that contains the small RNA (sRNA) and all the associated core proteins. Unexpectedly, this ribonucleoprotein is a homodimer, formed of two sRNAs and four copies of each of the core proteins. This dimer is likely to be the enzymatically active form, as mutations disrupting di-sRNP formation inhibited activity. Electron microscopy and single-particle analysis show that a small nuclear ribonucleoprotein forms a dimeric structure. Methylation of ribosomal RNA (rRNA) is required for optimal protein synthesis. Multiple 2′-O-ribose methylations are carried out by box C/D guide ribonucleoproteins [small ribonucleoproteins (sRNPs) and small nucleolar ribonucleoproteins (snoRNPs)], which are conserved from archaea to eukaryotes. Methylation is dictated by base pairing between the specific guide RNA component of the sRNP or snoRNP and the target rRNA. We determined the structure of a reconstituted and catalytically active box C/D sRNP from the archaeon Methanocaldococcus jannaschii by single-particle electron microscopy. We found that archaeal box C/D sRNPs unexpectedly formed a dimeric structure with an alternative organization of their RNA and protein components that challenges the conventional view of their architecture. Mutational analysis demonstrated that this di-sRNP structure was relevant for the enzymatic function of archaeal box C/D sRNPs.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is up-regulated in high-grade astrocytomas.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) controls the glycolytic flux via the allosteric activator fructose 2,6-bisphosphate. Because of its proto-oncogenic character, the PFK-2/FBPase-2 of the PFKFB3 gene is assumed to play a critical role in tumorigenesis. We investigated the PFKFB3 expression in 40 human astrocytic gliomas and 20 non-neoplastic brain tissue specimens. The PFKFB3 protein levels were markedly elevated in high-grade astrocytomas relative to low-grade astrocytomas and corresponding non-neoplastic brain tissue, whereas no significant increase of PFKFB3 mRNA was observed in high-grade astrocytomas when compared with control tissue. In the group of glioblastomas the PFKFB3 protein inversely correlates with EGFR expression. The findings demonstrate that PFKFB3 up-regulation is a hallmark of high-grade astrocytomas offering an explanation for high glycolytic flux and lactate production in these tumors.

Comprehensive mutational analysis of yeast DEXD/H box RNA helicases required for small ribosomal subunit synthesis.

ABSTRACT The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.

A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2–7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI: http://dx.doi.org/10.7554/eLife.00882.001

Mechanisms for Initiating Cellular DNA Replication

Diverse molecular choreography of replication Accurate duplication and transmission of genetic information to the next generation requires complex molecular assemblies. Bleichert et al. review replication initiation across the three domains of life, with a focus on origin selection and helicase loading. These processes identify potential origins of replication and prepare them for subsequent bidirectional replication initiation. There are key similarities and multiple differences in replication mechanisms between eukaryotes, prokaryotes, and archaea and many outstanding questions to be answered. Science, this issue p. eaah6317 BACKGROUND Cellular life depends on the ability of organisms to accurately duplicate and transmit genetic information between generations. In bacteria, archaea, and eukaryotes, this process uses sophisticated molecular machineries, termed replisomes, which copy chromosomal DNA through semiconservative replication. At the core of these replication factories reside ring-shaped hexameric helicases that assist polymerases in DNA synthesis by processively unzipping the parental DNA double helix. Replicative helicases are loaded onto DNA by dedicated initiator, loader, and accessory proteins during the initiation of DNA replication in a tightly regulated, multistep process. Although initiators and loaders are phylogenetically related between the bacterial and archaeal/eukaryotic lineages, the molecular choreography of DNA replication initiation turns out to be surprisingly diverse across different systems and employs different strategies to prepare replication origins for subsequent replisome assembly. ADVANCES Early work in bacterial systems established that initiator proteins recognize specific sequence elements (termed replication origins). Nucleotide-dependent oligomerization of the bacterial initiator DnaA at origins facilitates DNA melting and provides an access point for the replicative helicase, DnaB, which is recruited to and loaded onto each of the single-stranded origin regions by interactions with the initiator and other loader proteins to establish bidirectional replication forks. It has recently become clear that the strategies for origin recognition, helicase loading, and duplex DNA melting in archaeal and eukaryal systems, as well as the order of these steps, deviate considerably from the archetypal initiation events followed by bacteria. Although nucleotide-dependent interactions between initiator subunits still control early steps of replication initiation and regulate the association of initiators with origin DNA, they do not appear to contribute to DNA melting. Instead, archaeal/eukaryotic initiators [Orc in archaea or the multisubunit origin recognition complex (ORC) in eukaryotes] and helicase coloading factors cooperate to recruit and load the replicative helicase motor, the minichromosome maintenance (MCM) complex, onto duplex DNA in an inactive, double-hexameric form. Helicase activation, DNA melting, and replisome assembly do not occur until subsequent cell cycle phases, preventing premature replication and exposure of melted, single-stranded DNA regions to DNA damaging factors. OUTLOOK Although the core components of the initiation pathway have been identified, enabling reconstitution of the replication initiation cascade in vitro with recombinant proteins from bacterial and eukaryotic systems, the mechanisms by which these initiation factors function at the molecular level remain ill defined. Moreover, how replication initiation is temporally and spatially linked to the local chromatin environment and coordinated with other cellular (in bacteria and archaea) or nuclear (in eukaryotes) processes is not well understood. Future studies that integrate genetic, biochemical, biophysical, and structural approaches will be pivotal for resolving these questions. Overview of similarities and differences in the initiation of DNA replication across the three domains of life. Bacteria and archaea/eukaryotes share a requirement for an adenosine triphosphate (ATP)–dependent initiator factor that helps catalyze the loading of two ring-shaped, hexameric helicases onto a replication origin, thereby nucleating the formation of a bidirectional replication fork. The bacterial initiator forms a multimeric assembly that actively melts the origin before loading single helicase hexamers, whereas in archaea and eukaryotes, the helicase is loaded as an inactive dodecamer that then isomerizes into two active, single hexamers during or following origin melting. Cellular DNA replication factories depend on ring-shaped hexameric helicases to aid DNA synthesis by processively unzipping the parental DNA helix. Replicative helicases are loaded onto DNA by dedicated initiator, loader, and accessory proteins during the initiation of DNA replication in a tightly regulated, multistep process. We discuss here the molecular choreography of DNA replication initiation across the three domains of life, highlighting similarities and differences in the strategies used to deposit replicative helicases onto DNA and to melt the DNA helix in preparation for replisome assembly. Although initiators and loaders are phylogenetically related, the mechanisms they use for accomplishing similar tasks have diverged considerably and in an unpredictable manner.

Interdomain Communication of the Chd1 Chromatin Remodeler across the DNA Gyres of the Nucleosome.

Chromatin remodelers use a helicase-like ATPase motor to reposition and reorganize nucleosomes along genomic DNA. Yet, how the ATPase motor communicates with other remodeler domains in the context of the nucleosome has so far been elusive. Here, we report for the Chd1 remodeler a unique organization of domains on the nucleosome that reveals direct domain-domain communication. Site-specific cross-linking shows that the chromodomains and ATPase motor bind to adjacent SHL1 and SHL2 sites, respectively, on nucleosomal DNA and pack against the DNA-binding domain on DNA exiting the nucleosome. This domain arrangement spans the two DNA gyres of the nucleosome and bridges both ends of a wrapped, ∼90-bp nucleosomal loop of DNA, suggesting a means for nucleosome assembly. This architecture illustrates how Chd1 senses DNA outside the nucleosome core and provides a basis for nucleosome spacing and directional sliding away from transcription factor barriers.

Dissecting the role of conserved box C/D sRNA sequences in di-sRNP assembly and function

In all three kingdoms of life, nucleotides in ribosomal RNA (rRNA) are post-transcriptionally modified. One type of chemical modification is 2′-O-ribose methylation, which is, in eukaryotes and archaea, performed by box C/D small ribonucleoproteins (box C/D sRNPs in archaea) and box C/D small nucleolar ribonucleoproteins (box C/D snoRNPs in eukaryotes), respectively. Recently, the first structure of any catalytically active box C/D s(no)RNP determined by electron microscopy and single particle analysis surprisingly demonstrated that they are dimeric RNPs. Mutational analyses of the Nop5 protein interface suggested that di-sRNP formation is also required for the in vitro catalytic activity. We have now analyzed the functional relevance of the second interface, the sRNA interface, within the box C/D di-sRNP. Mutations in conserved sequence elements of the sRNA, which allow sRNP assembly but which severely interfere with the catalytic activity of box C/D sRNPs, prevent formation of the di-sRNP. In addition, we can observe the dimeric box C/D sRNP architecture with a different box C/D sRNP, suggesting that this architecture is conserved. Together, these results provide further support for the functional relevance of the di-sRNP architecture and also provide a structural explanation for the observed defects in catalysis of 2′-O-ribose methylation.

Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

Significance Stem cell factor (SCF) mediates its cellular responses by binding to and activating KIT, a transmembrane receptor tyrosine kinase. Here we describe an electron microscopy (EM) 3D reconstruction of negatively stained preparations of SCF-stimulated full-length KIT dimers. Assessment of the EM structure in respect to X-ray crystal structures of KIT extracellular and tyrosine kinase domains reveals the relative positioning of the individual domains in the context of the entire SCF-KIT complex. Whereas the homotypic contacts between the two KIT protomers show a consistent twofold symmetry for the ectodomains, the cytoplasmic arrangement is asymmetric and is found in several discrete conformations. The observed asymmetric contacts between tyrosine kinases may represent molecular interactions occurring during trans autophosphorylation and kinase stimulation. Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions in the membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions occurring during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT.