DNA topoisomerases, capable of manipulating DNA topology, are ubiquitous and indispensable for cellular survival due to the numerous roles they play during DNA metabolism. As we review here, current structural approaches have revealed unprecedented insights into the complex DNA‐topoisomerase interaction and strand passage mechanism, helping to advance our understanding of their activities in vivo. This has been complemented by single‐molecule techniques, which have facilitated the detailed dissection of the various topoisomerase reactions. Recent work has also revealed the importance of topoisomerase (...) interactions with accessory proteins and other DNA‐associated proteins, supporting the idea that they often function as part of multi‐enzyme assemblies in vivo. In addition, novel topoisomerases have been identified and explored, such as topo VIII and Mini‐A. These new findings are advancing our understanding of DNA‐related processes and the vital functions topos fulfil, demonstrating their indispensability in virtually every aspect of DNA metabolism. (shrink)

The assembly of nucleosomes and higher‐order chromatin structures has been extensively studied in vitro. Provided that non‐specific charge interactions are controlled, all the information for correct assembly is found to be inherent in the macromolecular components. Cellular extracts which can assemble chromatin in vitro with nucleosomes correctly spaced on the DNA have been studied in detail and also used to investigate the role of chromatin structure in transcription. However, the mechanisms of chromatin assembly in vivo are still (...) controversial. (shrink)

We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate (...) assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments. 10.1126/science.1151721. (shrink)

Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3′ primed DNA by the replication factor C (RFC) hetero‐pentameric clamp loader. Eukaryotes also have three alternative RFC‐like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24‐RFC (Rad17‐RFC in human) that loads a (...) 9‐1‐1 heterotrimer clamp onto a recessed 5′ end of DNA. Recent structural studies of Rad24‐RFC have discovered an unexpected 5′ DNA binding site on the outside of the clamp loader and reveal how a 5′ end can be utilized for loading the 9‐1‐1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5′ DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism. (shrink)

The developing vertebrate oocyte autonomously makes most of the components of the machinery for DNA and protein synthesis, as well as ‘maternal’ mRNAs, needed immediately after fertilization. However, specialized egg constituents such as yolk proteins, egg white, cholesterol, lipoproteins and egg coat substances, are formed in the liver, oviduct and perhaps in follicle cells. The assembly of the developing egg is therefore a fascinating example of division of labor among oocytes and extra‐oocyte tissues, the coordination of activities of the (...) latter being achieved by hormones. (shrink)

Eukaryotic DNA replication initiates at multiple origins. In early fly and frog embryos, chromosomal replication is very rapid and initiates without sequence specificity. Despite this apparent randomness, the spacing of these numerous initiation sites must be sufficiently regular for the genome to be completely replicated on time. Studies in various eukaryotes have revealed that there is a strict temporal separation of origin “licensing” prior to S phase and origin activation during S phase. This may suggest that replicon size must be (...) already established at the licensing stage. However, recent experiments suggest that a large excess of potential origins are assembled along chromatin during licensing. Thus, a regular replicon size may result from the selection of origins during S phase. We review single molecule analyses of origin activation and other experiments addressing this issue and their general significance for eukaryotic DNA replication. BioEssays 25:116–125, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

During early embryonic development in several metazoans, accurate DNA replication is ensured by high number of replication origins. This guarantees rapid genome duplication coordinated with fast cell divisions. In Xenopus laevis embryos this program switches to one with a lower number of origins at a developmental stage known as mid‐blastula transition (MBT) when cell cycle length increases and gene transcription starts. Consistent with this regulation, somatic nuclei replicate poorly when transferred to eggs, suggesting the existence of an epigenetic memory suppressing (...) replication assembly origins at all available sites. Recently, it was shown that histone H1 imposes a non‐permissive chromatin configuration preventing replication origin assembly on somatic nuclei. This somatic state can be erased by SSRP1, a subunit of the FACT complex. Here, we further develop the hypothesis that this novel form of epigenetic memory might impact on different areas of vertebrate biology going from nuclear reprogramming to cancer development. (shrink)

A recurrent theme in eukaryotic genome regulation stipulates that the properties of DNA are strongly influenced by the nucleoprotein complex into which it is assembled. Methylation of cytosine residues in vertebrate genomes has been implicated in influencing the assembly of locally repressive chromatin architecture. Current models suggest that covalent modification of DNA results in heritable, long‐term transcriptional silencing. In October of 2003, two manuscripts1,2 were published that challenge important aspects of this model, suggesting that modulation of both DNA methylation (...) itself, as well as the machinery implicated in its interpretation, are involved in acute gene regulation. BioEssays 26:217–220, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

Chromosomes are not only carriers of the genetic material, but also actively regulate the assembly of complex intracellular architectures. During mitosis, chromosome‐induced microtubule polymerisation ensures spindle assembly in cells without centrosomes and plays a supportive role in centrosome‐containing cells. Chromosomal signals also mediate post‐mitotic nuclear envelope (NE) re‐formation. Recent studies using novel approaches to manipulate histones in oocytes, where functions can be analysed in the absence of transcription, have established that nucleosomes, but not DNA alone, mediate the chromosomal (...) regulation of spindle assembly and NE formation. Both processes require the generation of RanGTP by RCC1 recruited to nucleosomes but nucleosomes also acquire cell cycle stage specific regulators, Aurora B in mitosis and ELYS, the initiator of nuclear pore complex assembly, at mitotic exit. Here, we review the mechanisms by which nucleosomes control assembly and functions of the spindle and the NE, and discuss their implications for genome maintenance. (shrink)

The information contained within the linear sequence of bases (the genome) must be faithfully replicated in each cell cycle, with a balance of constancy and variation taking place over the course of evolution. Recently, it has become clear that additional information important for genetic regulation is contained within the chromatin proteins associated with DNA (the epigenome). Epigenetic information also must be faithfully duplicated in each cell cycle, with a balance of constancy and variation taking place during the course of development (...) to achieve differentiation while maintaining identity within cell lineages. Both the genome and the epigenome are synthesized at the replication fork, so the events occurring during S‐phase provide a critical window of opportunity for eliciting change or maintaining existing genetic states. Cells discriminate between different states of chromatin through the activities of proteins that selectively modify the structure of chromatin. Several recent studies report the localization of certain chromatin modifying proteins to replication forks at specific times during S‐phase. Since transcriptionally active and inactive chromosome domains generally replicate at different times during S‐phase, this spatiotemporal regulation of chromatin assembly proteins may be an integral part of epigenetic inheritance. BioEssays 25:647–656, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

The hardest part of replicating a genome is the beginning. The first step of DNA replication (called “initiation”) mobilizes a large number of specialized proteins (“initiators”) that recognize specific sequences or structural motifs in the DNA, unwind the double helix, protect the exposed ssDNA, and recruit the enzymatic activities required for DNA synthesis, such as helicases, primases and polymerases. All of these components are orderly assembled before the first nucleotide can be incorporated. On the occasion of the 50th anniversary of (...) the discovery of the DNA structure, we review our current knowledge of the molecular mechanisms that control initiation of DNA replication in eukaryotic cells, with particular emphasis on the recent identification of novel initiator proteins. We speculate how these initiators assemble molecular machines capable of performing specific biochemical tasks, such as loading a ring‐shaped helicase onto the DNA double helix. BioEssays 25:1158–1167, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

Nucleosome positioning is proposed to have an essential role in facilitating the regulated transcription of eukaryotic genes. Some transcription factors can bind to DNA when it is appropriately wrapped around the histone core, others cannot bind due to the severe deformation of DNA structure. The staged assembly of nucleosomes and positioning of histone‐DNA contacts away from promoter elements can facilitate the access of transcription factors to DNA. Positioned nucleosomes can also facilitate transcription through providing the appropriate scaffolding to bring (...) regulatory factors bound at dispersed sites into juxtaposition. (shrink)

Nucleosome positioning is proposed to have an essential role in facilitating the regulated transcription of eukaryotic genes. Some transcription factors can bind to DNA when it is appropriately wrapped around the histone core, others cannot bind due to the severe deformation of DNA structure. The staged assembly of nucleosomes and positioning of histone‐DNA contacts away from promoter elements can facilitate the access of transcription factors to DNA. Positioned nucleosomes can also facilitate transcription through providing the appropriate scaffolding to bring (...) regulatory factors bound at dispersed sites into juxtaposition. (shrink)

Fundamental questions in evolution concern deep divisions in the living world and vertical versus horizontal information transfer. Two contrasting views are: (i) three superkingdoms Archaea, Eubacteria, and Eukarya based on vertical inheritance of genes encoding ribosomes; versus (ii) a prokaryotic/eukaryotic dichotomy with unconstrained horizontal gene transfer (HGT) among prokaryotes. Vertical inheritance implies continuity of cytoplasmic and structural information whereas HGT transfers only DNA. By hypothesis, HGT of the translation machinery is constrained by interaction between new ribosomal gene products and vertically (...) inherited cytoplasmic structure made largely of preexisting ribosomes. Ribosomes differentially enhance the assembly of new ribosomes made from closely related genes and inhibit the assembly of products from more distal genes. This hypothesis suggests experiments for synthetic biology: the ability of synthetic genomes to “boot,” i.e., establish hereditary continuity, will be constrained by the phylogenetic closeness of the cell “body” into which genomes are placed. (shrink)

A team of US researchers recently reported the design, assembly and in vivo functionality of a synthetic chromosome III (SynIII) for the yeast Saccharomyces cerevisiae. The synthetic chromosome was assembled bottom‐up from DNA oligomers by teams of students working over several years with researchers as the first part of an international synthetic yeast genome project. Embedded into the sequence of the synthetic chromosome are multiple design changes that include a novel in‐built recombination scheme that can be induced to catalyse (...) intra‐chromosomal rearrangements in a variety of different conditions. This system, along with the other synthetic sequence changes, is intended to aid researchers develop a deeper understanding of how genomes function and find new ways to exploit yeast in future biotechnologies. The landmark of the first synthesised designer eukaryote chromosome, and the power of its massively parallel recombination system, provide new perspectives on the future of synthetic biology and genome research. (shrink)

Markets, companies and various forms of business organizations may all be usefully viewed through the lens of CAS -- the theory of complex adaptive systems. In this chapter, I address one fundamental issue that confronts both the theoretician and the business manager: the nature and opportunities for control and intervention in complex adaptive regimes. The problem is obvious enough. A complex adaptive system, as we have defined it, is soft assembled and largely self-organizing. This means that it is the emergent (...) product of multiple, and often very heterogeneous, interacting factors and forces, and that the crucial interactions are not controlled and orchestrated by an overseeing executive, detailed program, or any other source of strict hierarchical control. There is thus a pressing problem -- are such systems strictly out of control and beyond the reach of useful governance? I shall argue that they are not. Such systems, though initially unfamiliar, can nonetheless be led, influenced and enabled in a variety of ways.I begin then, by clarifying the nature of the problem. What is it about complex adaptive systems that makes control and intervention so difficult? I then pursue a biological example: the role of genetic factors in influencing mature form. This example yields some lessons that can be applied to business organizations. Section 4 pursues a speculative extension of these lessons, applying them to the task of understanding the role of tagging and explicit models in certain kinds of advanced system. There is a brief conclusion. (shrink)

Alternative non‐B‐DNA conformations formed under physiological conditions by sequences called flipons include left‐handed Z‐DNA, three‐stranded triplexes, and four‐stranded i‐motifs and quadruplexes. These conformations accumulate and release energy to enable the local assembly of cellular machines in a context specific manner. In these transactions, nucleosomes store power, serving like rechargeable batteries, while flipons smooth energy flows from source to sink by acting as capacitors or resistors. Here, I review the known biological roles for flipons. I present recent and unequivocal findings (...) showing how innate immune responses are regulated by Z‐flipons that identify endogenous RNAs as self. Evidence is also presented supporting important roles for other flipon classes. In these examples, the dynamic exchange of energy between flipons and nucleosomes enables rapid switching of genetic programs without altering flipon sequence. The increased phenotypic diversity enabled by flipons drives their natural selection, with adaptations evolving faster than is possible by codon mutation alone. (shrink)

The premise of biological modularity is an ontological claim that appears to come out of practice. We understand that the biological world is modular because we can manipulate different parts of organisms in ways that would only work if there were discrete parts that were interchangeable. This is the foundation of the BioBrick assembly method widely used in synthetic biology. It is one of a number of methods that allows practitioners to construct and reconstruct biological pathways and devices using (...) DNA libraries of standardized parts with known functions. In this paper, we investigate how the practice of synthetic biology reconfigures biological understanding of the key concepts of modularity and evolvability. We illustrate how this practice approach takes engineering knowledge and uses it to try to understand biological organization by showing how the construction of functional parts and processes can be used in synthetic experimental evolution. We introduce a new approach within synthetic biology that uses the premise of a parts-based ontology together with that of organismal self-organization to optimize orthogonal metabolic pathways in E. coli. We then use this and other examples to help characterize semisynthetic categories of modularity, parthood, and evolvability within the discipline. (shrink)

The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG. CMG is “built” on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 “double hexamer.” In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel (...) in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins. Here, we review the structure and function of CMG, the 11 subunit helicase for eukaryotic DNA replication. Two CMGs are assembled at origins starting from two Mcm2-7 hexamers oriented N-to-N. The orientation of CMG at a forked DNA implies that the two CMGs at an origin pass one another. (shrink)

Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut‐shaped octamers with the potential to assemble into dimeric superstructures. The (...) presumed DNA‐binding mode suggests that RemA organizes its target DNA into nucleosome‐like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic‐stress adjustment. (shrink)

Several features of the yeast mitochondrial genome, including high mutation rate, dynamic genomic structure, small effective population size, and dispensability for cellular viability, make it a promising candidate for generating hybrid incompatibility and driving speciation. Cytonuclear incompatibility, a specific type of Dobzhansky‐Muller genetic incompatibility caused by improper interactions between mitochondrial and nuclear genomes, has previously been observed in a variety of organisms, yet its role in speciation remains obscure. Recent studies in Saccharomyces yeast species provide a new insight, with experimental (...) evidence that cytonuclear incompatibility and DNA sequence divergence are both causes of the reproductive isolation of different yeast species. Interestingly, these two mechanisms seem to be perfectly complementary to each other in terms of their effects and evolutionary trajectories. Direct molecular analyses of the incompatible genes in yeasts have started to shed light on the evolutionary forces driving speciation. Editor's suggested further reading in BioEssaysThe cytoplasmic structure hypothesis for ribosome assembly, vertical inheritance, and phylogeny AbstractMitochondrial bioenergetics as a major motive force of speciation Abstract. (shrink)

Kinetochores can form and be maintained on DNA sequences that are normally non‐centromeric. The existence of these so‐called neo‐centromeres has posed the problem as to the nature of the epigenetic mechanisms that maintain the centromere. Here we highlight results that indicate that the amount of CENP‐A at human centromeres is tightly regulated. It is also known that kinetochore assembly requires sister chromatid cohesion at mitosis. We therefore suggest that separation or stretching between the sister chromatids at metaphase reciprocally determines (...) the amount of centromere assembly in the subsequent interphase. This reciprocal relationship forms the basis of a negative feedback loop that could precisely control the amount of CENP‐A and faithfully maintain the presence of a kinetochore over many cell divisions. We describe how the feedback loop would work, propose how it could be tested experimentally and suggest possible components of its mechanism. (shrink)

Our aim in this article is to attempt to discuss propagating organization of process, a poorly articulated union of matter, energy, work, constraints and that vexed concept, “information”, which unite in far from equilibrium living physical systems. Our hope is to stimulate discussions by philosophers of biology and biologists to further clarify the concepts we discuss here. We place our discussion in the broad context of a “general biology”, properties that might well be found in life anywhere in the cosmos, (...) freed from the specific examples of terrestrial life after 3.8 billion years of evolution. By placing the discussion in this wider, if still hypothetical, context, we also try to place in context some of the extant discussion of information as intimately related to DNA, RNA and protein transcription and translation processes. While characteristic of current terrestrial life, there are no compelling grounds to suppose the same mechanisms would be involved in any life form able to evolve by heritable variation and natural selection. In turn, this allows us to discuss at least briefly, the focus of much of the philosophy of biology on population genetics, which, of course, assumes DNA, RNA, proteins, and other features of terrestrial life. Presumably, evolution by natural selection—and perhaps self-organization—could occur on many worlds via different causal mechanisms. Here we seek a non-reductionist explanation for the synthesis, accumulation, and propagation of information, work, and constraint, which we hope will provide some insight into both the biotic and abiotic universe, in terms of both molecular self reproduction and the basic work energy cycle where work is the constrained release of energy into a few degrees of freedom. The typical requirement for work itself is to construct those very constraints on the release of energy that then constitute further work. Information creation, we argue, arises in two ways: first information as natural selection assembling the very constraints on the release of energy that then constitutes work and the propagation of organization. Second, information in a more extended sense is “semiotic”, that is about the world or internal state of the organism and requires appropriate response. The idea is to combine ideas from biology, physics, and computer science, to formulate explanatory hypotheses on how information can be captured and rendered in the expected physical manifestation, which can then participate in the propagation of the organization of process in the expected biological work cycles to create the diversity in our observable biosphere. Our conclusions, to date, of this enquiry suggest a foundation which views information as the construction of constraints, which, in their physical manifestation, partially underlie the processes of evolution to dynamically determine the fitness of organisms within the context of a biotic universe. (shrink)

Error‐free genome duplication and accurate cell division are critical for cell survival. In all three domains of life, bacteria, archaea, and eukaryotes, initiator proteins bind replication origins in an ATP‐dependent manner, play critical roles in replisome assembly, and coordinate cell‐cycle regulation. We discuss how the eukaryotic initiator, Origin recognition complex (ORC), coordinates different events during the cell cycle. We propose that ORC is the maestro driving the orchestra to coordinately perform the musical pieces of replication, chromatin organization, and repair.

An astonishingly diverse biomolecular circuitry orchestrates the functioning machinery underlying every living cell. These biomolecules and their circuits have been engineered not only for various industrial applications but also to perform other atypical functions that they were not evolved for—including computation. Various kinds of computational challenges, such as solving NP‐complete problems with many variables, logical computation, neural network operations, and cryptography, have all been attempted through this unconventional computing paradigm. In this review, we highlight key experiments across three different ‘‘eras’’ (...) of molecular computation, beginning with molecular solutions, transitioning to logic circuits and ultimately, more complex molecular networks. We also discuss a variety of applications of molecular computation, from solving NP‐hard problems to self‐assembled nanostructures for delivering molecules, and provide a glimpse into the exciting potential that molecular computing holds for the future. Also see the video abstract here: https://youtu.be/9Mw0K0vCSQw. (shrink)

In Greg Bear’s critically acclaimed science fiction novel Darwin’s Radio, the activation of an endogenous retrovirus (SHEVA), ironically located in a “noncoding region” of the human genome, causes extreme symptoms in women worldwide, including miscarriages. In the United States, a task force is assembled to control the pandemic crisis and to find out how SHEVA operates at the genomic level. However, as the story unfolds, it becomes manifest that SHEVA is too complex to decode in this way and, moreover, that (...) it is not a disease at all. Biologist Kay Lang speculates that SHEVA is triggered by signals from the environment, and that newborn SHEVA children will be a new variation or species of Man. In this essay I analyze Bear’s literary experiment with science along Deleuze and Guattari’s important, but largely overlooked, concepts of State science and nomad science. Bear’s novel gives narrative form to nomad-scientific ideas about life, notably Lynn Margulis’s theory of endosymbiogenesis, which holds that a species’ DNA is an assemblage of many genomes acquired in symbiotic relations. The import of Bear’s informed speculations, I argue, is not crass prediction but a nomadic vision of life as always already different (impure, infected) and in becoming—a counterpoint to the image of the double helix as the bedrock of human identity. Darwin’s Radio is a key example of how fiction can be an excellent partner for science, technology, and society, analyzing and intervening in debates about life and laying bare epistemological and biopolitical tensions of technoscience. (shrink)

The central dogma of molecular biology dictates that, with only a few exceptions, information proceeds from DNA to protein through an RNA intermediate. Examining the enigmatic steps from prebiotic to biological chemistry, we take another road suggesting that primordial peptides acted as template for the self-assembly of the first nucleic acids polymers. Arguing in favour of a sort of archaic “reverse translation” from proteins to RNA, our basic premise is a Hadean Earth where key biomolecules such as amino acids, (...) polypeptides, purines, pyrimidines, nucleosides and nucleotides were available under different prebiotically plausible conditions, including meteorites delivery, shallow ponds and hydrothermal vents scenarios. Supporting a protein-first scenario alternative to the RNA world hypothesis, we propose the primeval occurrence of short two-dimensional peptides termed “selective amino acid- and nucleotide-matching oligopeptides” (henceforward SANMAOs) that noncovalently bind at the same time the polymerized amino acids and the single nucleotides dispersed in the prebiotic milieu. In this theoretical paper, we describe the chemical features of this hypothetical oligopeptide, its biological plausibility and its virtues from an evolutionary perspective. We provide a theoretical example of SANMAO’s selective pairing between amino acids and nucleosides, simulating a poly-Glycine peptide that acts as a template to build a purinic chain corresponding to the glycine’s extant triplet codon GGG. Further, we discuss how SANMAO might have endorsed the formation of low-fidelity RNA’s polymerized strains, well before the appearance of the accurate genetic material’s transmission ensured by the current translation apparatus. (shrink)

Changes in the abundance of protein and RNA molecules can impair the formation of complexes in the cell leading to toxicity and death. Here we exploit the information contained in protein, RNA and DNA interaction networks to provide a comprehensive view of the regulation layers controlling the concentration‐dependent formation of assemblies in the cell. We present the emerging concept that RNAs can act as scaffolds to promote the formation ribonucleoprotein complexes and coordinate the post‐transcriptional layer of gene regulation. We describe (...) the structural and interaction network properties that characterize the ability of protein and RNA molecules to interact and phase separate in liquid‐like compartments. Finally, we show that presence of structurally disordered regions in proteins correlate with the propensity to undergo liquid‐to‐solid phase transitions and cause human diseases. Also see the video abstract here https://youtu.be/kfpqibsNfS0. (shrink)

Contemporary textbooks often define evolution in terms of the replication, mutation, and selective retention of DNA sequences, ignoring the contribution of the physical processes involved. In the closing line of The Origin of Species, however, Darwin recognized that natural selection depends on prior more basic living functions, which he merely described as life’s “several powers.” For Darwin these involved the organism’s capacity to maintain itself and to reproduce offspring that preserve its critical functional organization. In modern terms we have come (...) to recognize that this involves the continual generation of complex organic molecules in complex configurations accomplished with the aid of persistent far-from-equilibrium chemical self-organizing and self-assembling processes. But reliable persistence and replication of these processes also requires constantly available constraints and boundary conditions. Organism autonomy further requires that these constraints and co-dependent dynamics are reciprocally produced, each by the other. In this paper I argue that the different constraint-amplifying dynamics of two or more self-organizing processes can be coupled so that they reciprocally generate each other’s critical supportive boundary conditions. This coupling is a higher-order constraint that effectively constitutes a sign vehicle “interpreted” by the synergistic dynamics of these co-dependent self-organizing process so that they reconstitute this same semiotic-dynamic relationship and its self-reconstituting potential in new substrates. This dynamical co-dependence constitutes Darwin’s “several powers” and is the basis of the biosemiosis that enables evolution. (shrink)

Minichromosome maintenance (Mcm) proteins are well‐known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS‐nonspecific group consisting of Mcm13, Mcm16‐19, and Mcm21‐22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high‐fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of (...) Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non‐overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure‐function of the ARS‐nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms. (shrink)

The conserved ribosomal protein uS3 in eukaryotes has long been known as one of the essential components of the small (40S) ribosomal subunit, which is involved in the structure of the 40S mRNA entry pore, ensuring the functioning of the 40S subunit during translation initiation. Besides, uS3, being outside the ribosome, is engaged in various cellular processes related to DNA repair, NF‐kB signaling pathway and regulation of apoptosis. This review is devoted to recent data opening new horizons in understanding the (...) roles of uS3 in such processes as the assembly and maturation of 40S subunits, ensuring proper structure of 48S pre‐initiation complexes, regulation of initiation and ribosome‐based RNA quality control pathways. Besides, we summarize novel results on the participation of the protein in processes beyond translation and consider biomedical implications of previously known and recently found extra‐ribosomal functions of uS3, primarily, in oncogenesis. (shrink)

The majority of G protein-coupled receptors (GPCRs) self-assemble in the form dimeric/oligomeric complexes along the plasma membrane. Due to the molecular interactions they participate, GPCRs can potentially provide the framework for discriminating a wide variety of intercellular signals, as based on some kind of combinatorial receptor codes. GPCRs can in fact transduce signals from the external milieu by modifying the activity of such intracellular proteins as adenylyl cyclases, phospholipases and ion channels via interactions with specific G-proteins. However, in spite of (...) the number of cell functions they can actually control, both GPCRs and their associated signal transduction pathways are extremely well conserved, for only a few alleles with null or minor functional alterations have so far been found. This would seem to suggest that, beside a mechanism for DNA repairing, there must be another level of quality control that may help maintaining GPCRs rather stable throughout evolution. We propose here receptor oligomerization to be a basic molecular mechanism controlling GPCRs redundancy in many different cell types, and the plasma membrane as the first hierarchical cell structure at which selective categorical sensing may occur. Categorical sensing can be seen as the cellular capacity for identifying and ordering complex patterns of mixed signals out of a contextual matrix, i.e., the recognition of meaningful patterns out of ubiquitous signals. In this context, redundancy and degeneracy may appear as the required feature to integrate the cell system into functional units of progressively higher hierarchical levels. (shrink)

The centromere is a specialized chromosomal structure that dictates kinetochore assembly and, thus, is essential for accurate chromosome segregation. Centromere identity is determined epigenetically by the presence of a centromere‐specific histone H3 variant, CENP‐A, that replaces canonical H3 in centromeric chromatin. Here, we discuss recent work by Roulland et al. that identifies structural elements of the nucleosome as essential determinants of centromere function. In particular, CENP‐A nucleosomes have flexible DNA ends due to the short αN helix of CENP‐A. The (...) higher flexibility of the DNA ends of centromeric nucleosomes impairs binding of linker histones H1, while it facilitates binding of other essential centromeric proteins, such as CENP‐C, and is required for mitotic fidelity. This work extends previous observations indicating that the differential structural properties of CENP‐A nucleosomes are on the basis of its contribution to centromere identity and function. Here, we discuss the implications of this work and the questions arising from it. (shrink)

Extensive regions of chromosomes can be transcriptionally repressed through silencing mechanisms mediated by complex chromatin structures. One of the most refined molecular portraits of silenced chromatin comes from studies of the silent mating‐type loci and telomeres of S. cerevisiae. In this budding yeast, the Sir3p silent information regulator emerges as a critically important silencing component that interacts with nucleosomes and other silencing proteins. Not only is it essential for silencing, but Sir3p is also capable of spreading silenced chromatin when its (...) dosage is increased. Sir3p is a target of mitogen‐activated protein (MAP) kinase cascade regulation and has significant similarity to the Orc1p subunit of the DNA replication origin recognition complex. Thus, in concert with other silencing proteins, Sir3p appears poised to respond to cellular signals and reprogram silencing through replication‐associated assembly of repressive chromatin structures. BioEssays 20:30–40, 1998. © 1998 John Wiley & Sons, Inc. -/- . (shrink)

Cancer cells accumulate widespread local and global chromatin changes and the source of this instability remains a key question. Here we hypothesize that chromatin alterations including unscheduled silencing can arise as a consequence of perturbed histone dynamics in response to replication stress. Chromatin organization is transiently disrupted during DNA replication and maintenance of epigenetic information thus relies on faithful restoration of chromatin on the new daughter strands. Acute replication stress challenges proper chromatin restoration by deregulating histone H3 lysine 9 mono‐methylation (...) on new histones and impairing parental histone recycling. This could facilitate stochastic epigenetic silencing by laying down repressive histone marks at sites of fork stalling. Deregulation of replication in response to oncogenes and other tumor‐promoting insults is recognized as a significant source of genome instability in cancer. We propose that replication stress not only presents a threat to genome stability, but also jeopardizes chromatin integrity and increases epigenetic plasticity during tumorigenesis. (shrink)

Homologous recombination (HR) is required to protect and restart stressed replication forks. Paradoxically, the Mrc1 branch of the S phase checkpoints, which is activated by replicative stress, prevents HR repair at breaks and arrested forks. Indeed, the mechanisms underlying HR can threaten genome integrity if not properly regulated. Thus, understanding how cells avoid genetic instability associated with replicative stress, a hallmark of cancer, is still a challenge. Here I discuss recent results that support a model by which HR responds to (...) replication stress through replicative and repair activities that operate at different stages of the cell cycle (S and G2, respectively) and in distinct subnuclear structures. Remarkably, the replication checkpoint appears to control this scenario by inhibiting the assembly of HR repair centers at stressed forks during S phase, thereby avoiding genetic instability. (shrink)

Introduction A Brief Conceptual Framework for Biology PART ONE: UNDERSTANDING NATURE 1. The Antecedents of Scientific Thought Animism, Totemism, and Shamanism The Paleolithic View Mesopotamia Egypt 2. Aristotle and the Greek View of Nature The Science of Animal Biology The Parts of Animals The Classification of Animals The Aristotelian System Basic Questions 3. Those Rational Greeks? Theophrastus and the Science of Botany The Roman Pliny Hippocrates, the Father of Medicine Erasistratus Galen of Pergamum The Greek Miracle 4. The Judeo-Christian Worldview (...) The Bishop of Hippo Scholastic Thought Islamic Science Books on Beasts Antecedents of a Revolution 5. The Revival of Science Andreas Vesalius and the Study of Structure William Harvey and the Study of Function Sir Francis Bacon’s Great Instauration Induction, Hypothesis, Deduction The Very Small--Animalcules Robert Hooke and the Discovery of Cells 6. Figur’d Stones and Plastick Virtue Marine Life on Mountain Tops? Figured Stones of Unknown Creatures Baron Cuvier Quarries of the Paris Basin Catastrophism and Uniformitarianism William Smith and the Geological Column Understanding Nature in 1850 PART TWO: THE GROWTH OF EVOLUTIONARY THOUGHT 7. The Paradigm of Evolution First Questions The Paradigm of Natural Theology First Answers 8. Testing Darwins Hypotheses Have Life Forms Changed over Time? Do Species Evolve into Different Species over Time? Has There Been Time Enough for Evolution? Is Natural Selection the Mechanism of Change? The Genetic Basis of Natural Selection Accounting for the Diversity of Life 9. In the Light of Evolution Comparative Anatomy Embryonic Development Classification Microstructure Molecular Processes 10. Life over Time The Origin of Life The Rise of Multicelled Organisms What Is a Phylum? Burgess Shale Metazoans Early Evolution of the Vertebrates The Age of Dinosaurs Birds, Mammals, and Flowering Plants The Rancho La Brea Tar Pits Human Evolution The Role of Extinction in Evolution PART THREE: CLASSICAL GENETICS 11. Pangenesis What Is the Question? Hippocrates and Aristotle The Darwinian Answer Assembling the Data Formulating the Hypothesis by Induction Galton’s Rabbits 12. The Cell Theory The Discovery of Cells: Robert Hooke Schwann and Cells in Animals Gametes as Cells Omnis cellula e cellula? The Technology of Cell Research 13. The Hypothesis of Chromosomal Continuity The Ephemeral Nucleus Schneider, Flemming, and Cell Division The Chromosomes and inheritance Gamete Formation Fertilization 14. Mendel and the Birth of Genetics Model for Monohybrid Crosses Model for Dihybrid Crosses Mendel’s Laws Initial Opposition to Mendelism 15. Genetics + Cytology: 1900-1910 Sutton’s Model The Cytological Basis of Mendel’s Laws Boveri and Abnormal Chromosome Sets Variations in Mendelian Ratios The Discovery of Sex Chromosomes 16. The Genetics of the Fruit Fly Morgan’s First Hypothesis Morgan’s Second Hypothesis The Fly Room Linkage and Crossing-Over The Cytological Proof of Crossing-Over Mapping the Chromosomes The Final Proof The Determinants of Sex The Conceptual Foundations of Classical Genetics 17. The Structure and Function of Genes One Gene, One Enzyme The Substance of Inheritance The Watson-Crick Model of DNA Genes and the Synthesis of Proteins The Genetic Code PART FOUR: THE ENIGMA OF DEVELOPMENT 18. First Principles The Peripatetic Stagirite The Death and Rebirth of Scientific Thought Harvey and Malpighi A Two-Millennial Summing Up Preformation versus Epigenesis 19. The Century of Discovery Von Baer’s Discovery of the Mammalian Ovum Darwin’s Contribution to Embryology Haeckel and Recapitulation 20. Descriptive Embryology Germ Layers The External Development of the Amphibian Embryo The Internal Development of the Amphibian Embryo 21. The Dawn of Analytical Embryology His, Roux, and Mosaic Development Driesch and Regulative Development Novelty in Development Cell Lineage Nucleus or Cytoplasm? Fin de Siècle 22. Interactions during Development Amphibian Organizers Secondary Organizers The Reacting Tissue The Chemical Nature of the Organizer Putting It All Together Conclusion Further Reading References Illustration Credits Index. (shrink)

Recent findings indicate that substantial cross‐talk may exist between transcriptional and post‐transcriptional processes. Firstly, there are suggestions that specific promoters influence the post‐transcriptional fate of transcripts, pointing to communication between protein complexes assembled on DNA and nascent pre‐mRNA. Secondly, an increasing number of proteins appear to be multifunctional, participating in transcriptional and post‐transcriptional events. The classic example is TFIIIA, required for both the transcription of 5S rRNA genes and the packaging of 5S rRNA. TFIIIA is now joined by the Y‐box (...) proteins, which bind DNA (transcription activation and repression) and RNA (mRNA packaging). Furthermore, the tumour suppressor WT1, at first thought to be a typical transcription factor, may also be involved in splicing; conversely, hnRNP K, a bona fide pre‐mRNA‐binding protein, appears to be a transcription factor. Other examples of multifunctional proteins are mentioned: notably PTB, Sxl, La and PU.1. It is now reasonable to assert that some proteins, which were first identified as transcription factors, could just as easily have been identified as splicing factors, hnRNP, mRNP proteins and vice versa. It is no longer appropriate to view gene expression as a series of compartmentalised processes; instead, multifunctional proteins are likely to co‐ordinate different steps of gene expression. (shrink)

The eukaryotic genome is packaged into a periodic nucleoprotein structure termed chromatin. The repeating unit of chromatin, the nucleosome, consists of DNA that is wound nearly two times around an octamer of histone proteins. To facilitate DNA‐directed processes in chromatin, it is often necessary to rearrange or to mobilize the nucleosomes. This remodeling of the nucleosomes is achieved by the action of chromatin‐remodeling complexes, which are a family of ATP‐dependent molecular machines. Chromatin‐remodeling factors share a related ATPase subunit and participate (...) in transcriptional regulation, DNA repair, homologous recombination and chromatin assembly. In this review, we provide an overview of chromatin‐remodeling enzymes and discuss two possible mechanisms by which these factors might act to reorganize nucleosome structure. BioEssays 25:1192–1200, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

The multistep pathway of eukaryotic gene expression involves a series of highly regulated events in the nucleus and cytoplasm. In the nucleus, genes are transcribed into pre‐messenger RNAs which undergo a series of nuclear processing steps. Mature mRNAs are then transported to the cytoplasm, where they are translated into protein and degraded at a rate dictated by transcript‐ and cell‐type‐specific cues. Until recently, these individual nuclear and cytoplasmic events were thought to be primarily regulated by different RNA‐ and DNA‐binding proteins (...) that are localized either only in the nucleus or only the cytoplasm. Here, we describe multifunctional proteins that control both nuclear and cytoplasmic steps of gene expression. One such class of multifunctional proteins (e.g., Bicoid and Y‐box proteins) regulates both transcription and translation whereas another class (e.g., Sex‐lethal) regulates both nuclear RNA processing and translation. Other events controlled by multifunctional proteins include assembly of spliceosome components, spliceosome recycling, RNA editing, cytoplasmic mRNA localization, and cytoplasmic RNA stability. The existence of multifunctional proteins may explain the paradoxical involvement of the nucleus in an RNA surveillance pathway (nonsense‐mediated decay) that detects cytoplasmic signals (premature termination codons). We speculate that shuttling multifunctional proteins serve to efficiently link RNA metabolism in the cytoplasmic and nuclear compartments. BioEssays 23:775–787, 2001. © 2001 John Wiley & Sons, Inc. (shrink)

Basic proteins and nucleic acids are assembled into complexes in a reaction that must be facilitated by nuclear chaperones in order to prevent protein aggregation and formation of non‐specific nucleoprotein complexes. The nucleophosmin/nucleoplasmin (NPM) family of chaperones [NPM1 (nucleophosmin), NPM2 (nucleoplasmin) and NPM3] have diverse functions in the cell and are ubiquitously represented throughout the animal kingdom. The importance of this family in cellular processes such as chromatin remodeling, genome stability, ribosome biogenesis, DNA duplication and transcriptional regulation has led to (...) the rapid growth of information available on their structure and function. The present review covers different aspects related to the structure, evolution and function of the NPM family. Emphasis is placed on the long‐term evolutionary mechanisms leading to the functional diversification of the family members, their role as chaperones (particularly as it pertains to their ability to aid in the reprogramming of chromatin), and the importance of NPM2 as an essential component of the amphibian chromatin remodeling machinery during fertilization and early embryonic development. BioEssays 29: 49–59, 2007. © 2006 Wiley Periodicals, Inc. (shrink)

Consistent with the complexity of the temporally regulated processes that must occur for growth and development of higher eukaryotes, it is now apparent that transcription is regulated by the formation of multi‐component complexes that assemble on the promoters of genes. These complexes can include (in addition to the five or more general transcription factors and RNA polymerase II) DNA‐binding proteins, transcriptional activators, coactivators, adaptors and various accessory proteins. The best studied example of a complex that includes a transcriptional adaptor, accessory (...) proteins and a DNA‐binding protein is that involving the herpes simplex virus VP16 protein. Evidence suggests that the adenovirus E1a protein and the cellular Sp1 and CTF/NF1 transcription factors also function through adaptors or coactivators. Each additional component of the transcription complex provides the cell with another point at which to exert control of gene expression. (shrink)

The glucocorticoid receptor belongs to an important class of transcription factors that alter the expression of target genes in response to a specific hormone signal. The glucocorticoid receptor can function at least at three levels: (1) recruitment of the general transcription machinery; (2) modulation of transcription factor action, independent of DNA binding, through direct protein‐protein interactions; and (3) modulation of chromatin structure to allow the assembly of other gene regulatory proteins and/or the general transcription machinery on the DNA. This (...) review will focus on the multifaceted nature of protein‐protein interactions involving the glucocorticoid receptor and basal transcription factors, coactivators and other transcription factors, occurring at these different levels of regulation. (shrink)

Just as the faithful replication of DNA is an essential process for the cell, chromatin structures of active and inactive genes have to be copied accurately. Under certain circumstances, however, the activity pattern has to be changed in specific ways. Although analysis of specific aspects of these complex processes, by means of model systems, has led to their further elucidation, the mechanisms of chromatin replication in vivo are still controversial and far from being understood completely. Progress has been achieved in (...) understanding: 1. The initiation of chromatin replication, indicating that a nucleosome‐free origin is necessary for the initiation of replication; 2. The segregation of the parental nucleosomes, where convincing data support the model of random distribution of the parental nucleosomes to the daughter strands; and 3. The assembly of histones on the newly synthesized strands, where growing evidence is emerging for a two‐step mechanism of nucleosome assembly, starting with the deposition of H3/H4 tetramers onto the DNA, followed by H2A/H2B dimers. (shrink)

Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere (...) DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in tethe cell cycle. (shrink)

Synthetic genomics is a new field of research in which small DNA pieces are assembled in a series of steps into whole genomes. The highly reduced genomes of host‐associated bacteria are now being used as models for de novo synthesis of small genomes in the laboratory. Bacteria with the smallest genomes identified in nature provide nutrients to their hosts, such as amino acids, co‐factors and vitamins. Comparative genomics of these bacteria enables predictions to be made about the gene sets required (...) for core cellular functions and the associated metabolic network for the biosynthesis of host‐selected compounds. Synthetic biology may ultimately enable researchers to make customized cell‐specific organelles for the production and delivery of drugs to humans and domestic animals. Synthetic genomics may also become the method of choice for functional analyses of genes and genomes from bacteria that cannot be cultivated in the laboratory. (shrink)

Culture-independent high-throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth’s most ubiquitous and diverse inhabitants – the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non-viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently (...) labeled viruses to their host cells via high-throughput flow sorting. Sequencing of such isolated virus–host pairs facilitates cultivation-independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts. (shrink)

We present a molecular model of eukaryotic gene transcription. For the β‐globin locus, we hypothesise that a transcription machine composed of multiple RNA polymerase II (PolII) assembles using the locus control region as a foundation. Transcription and locus remodelling can be achieved by pulling DNA through this multi‐PolII ‘reading head’. Once a transcription complex is formed, it may engage an active gene in several rounds of transcription. Observed intergenic sense and antisense transcripts may be the result of PolII pulling the (...) DNA through the reading head whilst searching for the promoter of a gene. Support for this hypothesis is provided using various data from the literature. In the model, DNA is packed in a 30‐nm chromatin fibre, thus gene regulatory regions separated by kilobases are close in space. This, and the need to store transcription‐induced supercoiling, may explain why functionally interacting regions are often separated by many kilobases. (shrink)

We present a molecular model of eukaryotic gene transcription. For the β‐globin locus, we hypothesise that a transcription machine composed of multiple RNA polymerase II (PolII) assembles using the locus control region as a foundation. Transcription and locus remodelling can be achieved by pulling DNA through this multi‐PolII ‘reading head’. Once a transcription complex is formed, it may engage an active gene in several rounds of transcription. Observed intergenic sense and antisense transcripts may be the result of PolII pulling the (...) DNA through the reading head whilst searching for the promoter of a gene. Support for this hypothesis is provided using various data from the literature. In the model, DNA is packed in a 30‐nm chromatin fibre, thus gene regulatory regions separated by kilobases are close in space. This, and the need to store transcription‐induced supercoiling, may explain why functionally interacting regions are often separated by many kilobases. (shrink)

Engineered microbes are of great potential utility in biotechnology and basic research. In principle, a cell can be built from scratch by assembling small molecule sets with auto‐catalytic properties. Alternatively, DNA can be isolated or directly synthesized and molded into a synthetic genome using existing genomic blueprints and molecular biology tools. Activating such a synthetic genome will yield a synthetic cell. Here we examine obstacles associated with this latter approach using a model system whereby a donor genome from H. influenzae (...) is fragmented, and the pieces are then modified and reassembled stepwise in an E. coli host cell. There are obstacles associated with this strategy related to DNA transfer, DNA replication, cross‐talk in gene regulation and compatibility of gene products between donor and host. Encouragingly, analysis of gene expression indicates widespread transcription of H. influenzae genes in E. coli, and analysis of gap locations in H. influenzae and other microbial genome assemblies reveals few genes routinely incompatible with E. coli. In conclusion, rebuilding and booting a genome remains a feasible and pragmatic approach to creating a synthetic microbial cell. BioEssays 29:580–590, 2007. © 2007 Wiley Periodicals, Inc. (shrink)