Many enzymes are inhibited by their own substrates, leading to velocity curves that rise to a maximum and then descend as the substrate concentration increases. Substrate inhibition is often regarded as a biochemical oddity and experimental annoyance. We show, using several case studies, that substrate inhibition often has important biological functions. In each case we discuss, the biological significance is different. Substrate inhibition of tyrosine hydroxylase results in a steady synthesis of dopamine despite large fluctuations in (...) tyrosine due to meals. Substrate inhibition of acetylcholinesterase enhances the neural signal and allows rapid signal termination. Substrate inhibition of phosphofructokinase ensures that resources are not devoted to manufacturing ATP when it is plentiful. In folate metabolism, substrate inhibition maintains reactions rates in the face of substantial folate deprivation. Substrate inhibition of DNA methyltransferase serves to faithfully copy DNA methylation patterns when cells divide while preventing de novo methylation of methyl‐free promoter regions. (shrink)

After yeast cells commit to the cell cycle in a process called START, genes required for DNA synthesis are expressed in late G1. Periodicity is mediated by a hexameric sequence, known as a MCB element, present in all DNA synthesis gene promoters. A complex that specifically binds MCBs has been identified. One polypeptide in the MCB complex is Swi6, a transcription factor that together with Swi4 also binds G1 cyclin promoters and participates in a positive feedback loop at (...) START. The finding that Swi6 is directly involved in both START and DNA synthesis gene control suggest a model in which Swi6, activated through its participation in START, serves as the central transcription factor in coordinating late G1 gene expression. The mechanism may be conserved in all eukaryotic cells. (shrink)

DNA replication stress threatens ordinary DNA synthesis. The evolutionarily conserved DNA replication stress response pathway involves sensor kinase Mec1/ATR, adaptor protein Mrc1/Claspin, and effector kinase Rad53/Chk1, which spurs a host of changes to stabilize replication forks and maintain genome integrity. DNA replication forks consist of largely distinct sets of proteins at leading and lagging strands that function autonomously in DNA synthesis in vitro. In this article, we discuss eSPAN and BrdU‐IP‐ssSeq, strand‐specific sequencing technologies that permit analysis of protein (...) localization and DNA synthesis at individual strands in budding yeast. Using these approaches, we show that under replication stress Rad53 stalls DNA synthesis on both leading and lagging strands. On lagging strands, it stimulates PCNA unloading, and on leading strands, it attenuates the replication function of Mrc1‐Tof1. We propose that in doing so, Rad53 couples leading and lagging strand DNA synthesis during replication stress, thereby preventing the emergence of harmful ssDNA. (shrink)

Adaptation by means of natural selection depends on the ability of populations to maintain variation in heritable traits. According to the Modern Synthesis this variation is sustained by mutations and genetic drift. Epigenetics, evodevo, niche construction and cultural factors have more recently been shown to contribute to heritable variation, however, leading an increasing number of biologists to call for an extended view of speciation and evolution. An additional common feature across the animal kingdom is learning, defined as the ability (...) to change behavior according to novel experiences or skills. Learning constitutes an additional source for phenotypic variation, and change in behavior may induce long lasting shifts in fitness, and hence favor evolutionary novelties. Based on published studies, I demonstrate how learning about food, mate choice and habitats has contributed substantially to speciation in the canonical story of Darwin’s finches on the Galapagos Islands. Learning cannot be reduced to genetics, because it demands decisions, which requires a subject. Evolutionary novelties may hence emerge both from shifts in allelic frequencies and from shifts in learned, subject driven behavior. The existence of two principally different sources of variation also prevents the Modern Synthesis from self-referring explanations. (shrink)

Dependency relationships within the cell cycle allow cells to arrest the cycle reversibly in response to agents or conditions that interfere with specific aspects of its normal progression. In addition, overlapping pathways exist which also arrest the cell cycle in response to DNA damage. Collectively, these control mechanisms have become known as checkpoints. Analysis of checkpoints is facilitated by the fact that dependency relationships within the cell cycle, such as the dependency of mitosis on the completion of DNA synthesis, (...) and the DNA damage checkpoint can be separated genetically. In fission yeast, Schizosaccharomyces pombe, the dependency of mitosis on prior completion of DNA synthesis is mediated through tyrosine‐15 phosphorylation of the ubiquitous mitotic regulator p34cdc2. In contrast, the arrest of mitosis caused by DNA damage acts through a separate mechanism that appears to be independent of tyrosine‐15 phosphorylation. Despite these distinct interactions with the mitotic machinery, the majority of fission yeast mutants that are deficient in mitotic arrest after DNA damage are also unable to respond to inhibition of DNA synthesis. In this essay we survey the current knowledge concerning feedback controls and checkpoints within fission yeast and relate this to information derived from other systems. (shrink)

Three major aspects of G1 regulation acting at START in fission yeast are discussed in this review. Firstly, progression towards S phase in the mitotic cycle. This is controlled by the activation of transcription complexes at START which cause cell cycle‐dependent activation of genes required for DNA synthesis. The second aspect is the regulation of developmental fate occurring during G1. Passage through START appears to inhibit sexual differentiation because the meiotic and mitotic pathways are mutually exclusive. This is brought (...) about because the meiotic pathway is inhibited by the same gene functions that are required for S phase onset. Thirdly, distinct checkpoint, or dependency, controls operate both pre‐ and post‐START in the mitotic cycle to inhibit mitosis in the absence of replicated DNA, and also to limit rounds of DNA replication to one per cell cycle. (shrink)

The vestibulo-ocular reflex (VOR) adaptation is an ideal model for investigating how the neurosteroid 17 beta-estradiol (E2) contributes to the modification of behavior by regulating synaptic activities. We hypothesized that E2 impacts VOR adaptation by affecting cerebellar synaptic plasticity at the parallel fiber–Purkinje cell (PF) synapse. To verify this hypothesis, we investigated the acute effect of blocking E2 synthesis on gain increases and decreases in adaptation of the VOR in male rats using an oral dose (2.5 mg/kg) of the (...) aromatase inhibitor letrozole. We also assessed the effect of letrozole on synaptic plasticity at the PF synapse in vitro, using cerebellar slices from male rats. We found that letrozole acutely impaired both gain increases and decreases adaptation of the VOR without altering basal ocular-motor performance. Moreover, letrozole prevented long-term potentiation at the PF synapse (PF-LTP) without affecting long-term depression (PF-LTD). Thus, in male rats neurosteroid E2 has a relevant impact on VOR adaptation and affects exclusively PF-LTP. These findings suggest that E2 might regulate changes in VOR adaptation by acting locally on cerebellar and extra-cerebellar synaptic plasticity sites. (shrink)

Werner syndrome (WS) is an inherited disorder that produces somatic stunting, premature ageing and early onset of degenerative and neoplastic diseases. Cultured fibroblasts derived from subjects with WS are found to undergo premature replicative senescence and thus provide a cellular model system to study the disorder. Recently, several overexpressed gene sequences isolated from a WS fibroblast cDNA library have been shown to possess the capacity to inhibit DNA synthesis and disrupt many normal biochemical processes. Because a similar constellation of (...) genes is overexpressed in WS and senescent normal fibroblasts, these data suggest the existence of a common molecular genetic pathway for replicative senescence in both types of cell. We propose that the primary defect in WS is a mutation in a gene for a trans‐acting repressor protein that reduces its binding affinity for shared regulatory regions of several genes, including those that encode inhibitors of DNA synthesis (IDS). The mutant WS repressor triggers a sequence of premature expression of IDS and other genes, with resulting inhibition of DNA synthesis and early cellular senescence, events which occur much later in normal cells. (shrink)

DNA gyrase, an enzyme unique to prokaryotes, has been implicated in almost all processes that involve DNA. Although efficient inhibitors of this protein have been known for more than 20 years, none of them have enjoyed prolonged pharmaceutical success. It is only recently that the mechanisms of inhibition for some of these classes of drugs have been established unequivocally by X‐ray crystallography. It is hoped that this detailed structural information will assist the design of novel, effective inhibitors of DNA (...) gyrase. (shrink)

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. In addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. mRNAs for both subunits are cell cycle regulated and inducible by DNA damage in all organisms examined, including E. coli, S. cerevisiae and H. sapiens. This DNA damage regulation is thought to provide a metabolic state that facilitates DNA replicational (...) repair processes. S. cerevisiae also encodes a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. Genetic analysis of the DNA damage response in S. cerevisiae has shown that RNR expression is under both positive and negative control. Among mutants constitutive for RNR expression are the general transcriptional repression genes, SSN6 and TUP1. Mutations in POL1 and POL3 also activate RNR expression, indicating that the DNA damage sensory network may respond directly to blocks in DNA synthesis. A protein kinase, Dun1, has been identified that controls inducibility of RNR1, RNR2 and RNR3 in response to DNA damage and replication blocks. This result suggests that the RNR genes in S. cerevisiae form a regulon that is coordinately regulated by protein phosphorylation in response to DNA damage. (shrink)

The synthesis and integration of DNA into the genome of its host cell is a normal step in the replication of the retroviruses. Previous studies have provided details concerning the structure of viral DNA and viral and host integration sites. More recent genetic and biochemical results have expanded our understanding considerably: it should soon be possible to describe the exact viral DNA sequence recognized during the integration reaction for several viruses. In addition, at least one of the viral proteins (...) and enzymatic activities required in the reaction has been identified. Analysis of this apparently efficient and highly specific site‐directed recombination event in eukaryotic cells promises to provide insights of both fundamental and practical interest. (shrink)

A science is an intellectual activity defined by its mechanisms that prevent its scientists from always reaching the conclusions that they set out to reach. Such mechanisms are needed because, if scientists are given full control over what hypotheses they select, what data they discard, and what results they publish, they can communicate any conclusion that they desire. Synthesis, by setting a grand challenge, forces scientists across uncharted territory where they encounter and solve unscripted problems. When theory is inadequate, (...) the synthesis fails, and fails in a way that cannot be ignored. Therefore, synthesis drives discovery and paradigm change in ways that simple hypothesis testing cannot. Here, we describe the discoveries that emerged when synthetic biologists were challenged to create an alternative genetic system that has different molecular structures than DNA and RNA. In pursuit of this particular “grand challenge,” synthesis forced the recognition that the community did not know as much about the double helix as it had constructively assumed. In general, a field of science having access to synthesis as a research strategy can create knowledge even if its practitioners do not fit the ideal of a dispassionate, advocacy-free scientist. (shrink)

There is growing evidence that mutations can arise in non‐dividing cells (both bacterial and mammalian) in the absence of chromosomal replication. The processes that are involved are still largely unknown but may include two separate mechanisms. In the first, DNA lesions resulting from the action of endogenous mutagens may give rise to RNA transcripts with miscoded bases. If these confer the ability to initiate DNA replication, the DNA lesions may have an opportunity to miscode during replication and thus could give (...) rise to apparently ‘adaptive’ mutations. A second mechanism is suggested by recent work in starved bacteria, showing that there is much more turnover of chromosomal DNA than has been previously thought. This could permit polymerase errors to lead to mutations in non‐dividing cells. Such cryptic DNA synthesis, which may essentially replace existing DNA rather than duplicating it, could, in principle, act as an additional source of variability on which selection may act, initially in the absence of cell division. In mammals such processes would undoubtedly have implications for germ cell mutagenesis and carcinogenesis. (shrink)

The model of DNA-histones has the following elements: The hydrogen bonds between the complementary nucleotide bases function as informational gates. When the electrons π of one nucleotide base are excited, an exchange of protons is produced between the two complementary bases. The result is the displacement of the conjugated double bonds which facilitates the inter-molecular transmission of the electronic wave of excitation by electro-magnetic coupling. Each triplet of nucleotide bases of DNA fixes one definite amino acid . Between the nucleotide (...) bases and the amino acids there are constituted informational gates, which ensure the circulation of the electronic wave of excitation. An input signal molecule arrives at the receiver gene and unleashes the activity of the enzymes which introduce in the DNA-histones system the electronic wave of excitation. The electronic wave of excitation arises as a result of the break of the high-energy bonds of ATP. Then, the electronic excitation is transmitted to the productor gene where it represents the signal for starting the synthesis of the mRNA.Le modèle de système ADN-Histone a les éléments suivants: Les liaisons d'hydrogène entre les bases nucléotidiques complémentaires fonctionnent comme des portes informationnelles. Quand les électrons π d'une base nucléotidique sont excités, il se produit un échange de protons entre les bases complémentaires. Le résultat est un déplacement des doubles liaisons conjugées qui facilite la transmission de l'onde d'excitation électronique par un couplage électromagnétique. Chaque triplet de bases nucléotidiques de l'ADN fixe un certain amino-acide . Entre les bases nucléodiques et les amino-acides se constituent des portes informationnelles qui assurent la circulation de l'onde d'excitation dlectronique. Une molécule signal d'entrée arrive au gène récepteur et déclenche l'activité des enzymes qui introduisent dans le système ADN-histone l'excitation électronique. L'excitation électronique apparait comme résultat de la rupture des liaisons de haute énergie del' ATP. l'Onde d'excitation électronique est transmise au gène producteur, où elle rdprésente le signal pour faire commencer la synthèse de l'ARNm.Das Modell des DNS-Histonen Systems weist folgende Elemente auf: Die Wasserstoffbindungen zwischen den komplementäaren Nukleotidbasen funktionieren wie informationale Pforten. Wenn die π Elektronen einer Nukleotidbase erregt werden, ensteht ein Protonenwechsel zwischen den Komplementären Basen. Das Ergebnis ist eine Verschiebung der konjugierten Doppelbindungen, die die Weiterleitung der elektronischen Erregungswelle durch eine elektromagnetische Kupplung erleichtert. Jedes Nukleotidbasentriplett der DNS fixiert eine bestimmte Aminosäure . Zwischen den Nukelotidbasen und den Aminosäuren entstehen Informationspforten, welche den Umlauf der elektronischen Erregungswelle sichern. Ein Eintrittsignal Moleküul gelangt zur Rezeptorgen und löst die Aktivität von Enzymen aus, welche die elektronische Erregungswelle in das DNS-Histonen System einführen werden. Die elektronische Erregungswelle erscheint als Ergebnis der Spaltung der hochenergetischen Bindungen des ATP-s. Die elektronische Erregungswelle wird alsdann zur Produktorgen weitergeleitet, wo sie das Signal zum Beginn der Synthese der mRNS darstellt. (shrink)

Oxidative damage to DNA appears to be a factor in cancer, yet explanations for why highly elevated levels of such lesions do not always result in cancer remain elusive. Much of the genome is non‐coding and lesions in these regions might be expected to have little biological effect, an inference supported by observations that there is preferential repair of coding sequences. RNA has an important coding function in protein synthesis, and yet the consequences of RNA oxidation are largely unknown. (...) Some non‐coding nucleic acid is functional, e.g. promoters, and damage to these sequences may well have biological consequences. Similarly, oxidative damage to DNA may promote microsatellite instability, inhibit methylation and accelerate telomere shortening. DNA repair appears pivotal to the maintenance of genome integrity, and genetic alterations in repair capacity, due to single nucleotide polymorphisms or mutation, may account for inter‐individual differences in cancer susceptibility. This review will survey these aspects of oxidative damage to nucleic acids and their implication for disease. BioEssays 26:533–542, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3′→5′ exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is (...) that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV‐damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication. (shrink)

While large portions of the mammalian genome are known to replicate sequentially in a distinct, tissue‐specific order, recent studies suggest that the inactive X chromosome is duplicated rapidly via random, synchronous DNA synthesis at numerous adjacent regions. The rapid duplication of the inactive X chromosome was observed in high‐resolution studies visualizing DNA replication patterns in the nucleus, and by allele‐specific DNA sequencing studies measuring the extent of DNA synthesis. These studies conclude that inactive X chromosomes complete replication earlier (...) than previously thought and suggest that the strict order of DNA replication detected in the majority of genomic regions is not preserved in non‐transcribed, “silent” chromatin. These observations alter current concepts about the regulation of DNA replication in non‐transcribed portions of the genome in general and in the inactive X‐chromosome in particular. (shrink)

The many genetic complementation groups of DNA excision‐repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell (...) extracts and is defective in extracts from cell lines derived from individuals with the excisionrepair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single‐stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication. (shrink)

The short paper introduces the concept of possible branches of double-stranded DNA (later sometimes called palindromes): Certain sequences of nucleotides may be followed, after a short unpaired stretch, by a complementary sequence in reversed order, such that each DNA strand can fold back on itself, and the DNA assumes a cruciform or tree-like structure. This is postulated to interact with regulatory proteins. -/- .

This paper assesses Sarkar's ([2003]) deflationary account of genetic information. On Sarkar's account, genes carry information about proteins because protein synthesis exemplifies what Sarkar calls a ‘formal information system’. Furthermore, genes are informationally privileged over non-genetic factors of development because only genes enter into arbitrary relations to their products (in virtue of the alleged arbitrariness of the genetic code). I argue that the deflationary theory does not capture four essential features of the ordinary concept of genetic information: intentionality, exclusiveness, (...) asymmetry, and causal relevance. It is therefore further removed from what is customarily meant by genetic information than Sarkar admits. Moreover, I argue that it is questionable whether the account succeeds in demonstrating that information is theoretically useful in molecular genetics. Introduction Sarkar's Information System The Pre-theoretic Features of Genetic Information 3.1 Intentionality 3.2 Exclusiveness 3.3 Asymmetry 3.4 Causal relevance Theoretical Usefulness Conclusion CiteULike Connotea Del.icio.us What's this? (shrink)

DNA methylation is a quintessential epigenetic mechanism. Widely considered a stable regulator of gene silencing, it represents a form of “molecular braille,” chemically printed on DNA to regulate its structure and the expression of genetic information. However, there was a time when methyl groups simply existed in cells, mysteriously speckled across the cytosine building blocks of DNA. Why was the code of life chemically modified, apparently by “no accident of enzyme action” (Wyatt 1951 )? If all cells in a body (...) share the same genome sequence, how do they adopt unique functions and maintain stable developmental states? Do cells remember? In this historical perspective, I review epigenetic history and principles and the tools, key scientists, and concepts that brought us the synthesis and discovery of prokaryotic and eukaryotic methylated DNA. Drawing heavily on Gerard Wyatt’s observation of asymmetric levels of methylated DNA across species, as well as to a pair of visionary 1975 DNA methylation papers, 5-methylcytosine is connected to DNA methylating enzymes in bacteria, the maintenance of stable cellular states over development, and to the regulation of gene expression through protein-DNA binding. These works have not only shaped our views on heritability and gene regulation but also remind us that core epigenetic concepts emerged from the intrinsic requirement for epigenetic mechanisms to exist. Driven by observations across prokaryotic and eukaryotic worlds, epigenetic systems function to access and interpret genetic information across all forms of life. Collectively, these works offer many guiding principles for our epigenetic understanding for today, and for the next generation of epigenetic inquiry in a postgenomics world. (shrink)

During the past few years significant progress has been made in our understanding of the structure and function of the proteins involved in eukaryotic DNA replication. Data from several laboratories suggest that, in contrast to prokaryotic DNA replication, two distinct DNA polymerases are required for eukaryotic DNA replication, i.e. DNA polymerase delta for the synthesis of the leading strand and DNA polymerase alpha for the lagging strand. Several accessory proteins analogous to prokaryotic replication factors have been identified and some (...) of these are specific for pol delta whereas others affect both DNA replicases. The replicases and their accessory proteins appear to be highly conserved in eukaryotes, as homologous proteins have been found in species ranging from humans to yeast. (shrink)

Under certain conditions, the cell cycle can be arrested for a long period of time. Vertebrate oocytes are arrested at G2 phase, while somatic cells arrest at G0 phase. In both cells, nuclei have lost the ability to initiate DNA synthesis. In a pair of recently published papers,1,2 Méchali and colleagues and Coué and colleagues have clarified how frog oocytes prevent untimely DNA synthesis during the long G2 arrest. Intriguingly, they found only Cdc6 is responsible for the inability (...) of immature oocytes to replicate DNA. Cdc6 is a key component for replication licensing, and for G0 cells to re‐enter the proliferative stage. Strikingly similar strategies for preventing the untimely replication in both cells suggest that the suppression of replication licensing is a universal mechanism for securing the prolonged arrest of the cell cycle. BioEssays 25:313–316, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

Certain sequences of double‐helical DNA can be recognized and tightly bound by oligonucleotides. The effects of such triple‐helical structures on DNA binding proteins have been studied. Stabilities of DNA triple‐helices at or near physiological conditions are sufficient to inhibit DNA binding proteins directed to overlapping sites. Such proteins include restriction endonucleases, methylases, transcription factors, and RNA polymerases. These and Other results suggest that oligonucleotide‐directed triple‐helix formation could provide the basis for designing artificial gene repressors. The general question of whether biological (...) systems employ RNA molecules for recognition and regulation of double‐helical DNA is discussed. (shrink)

The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in all organisms analysed to date. In human cells, representatives of this family include the proteins defective in the cancer predisposition disorder Bloom's syndrome and the premature ageing condition, Werner's syndrome. Several pieces of evidence suggest that RecQ family helicases form associations with one or more of the cellular topoisomerases, and together these heteromeric complexes manipulate DNA structure to effect efficient DNA replication, (...) genetic recombination, or both. Here, we propose that RecQ helicases are required for ensuring that structural abnormalities arising during replication, such as at sites where replication forks encounter DNA lesions, are corrected with high fidelity. In mutants defective in these proteins, not only is replication abnormal, but cells display aberrant responses to DNA-damaging agents or inhibitors of DNA synthesis. We suggest that RecQ helicases may be important for the integration of cellular responses to these insults, such as by linking cell cycle checkpoint responses to recombinational repair. BioEssays 21:286–294, 1999. © 1999 John Wiley & Sons, Inc. (shrink)

Besides its use in basic research, the DNA:RNA hybridization technique has helped the development of genetic engineering: it is instrumental in the isolation of specific genes that can be inserted into foreign cells, thus modifying their genetic information. Plants, animals, and microorganisms can now be altered to yield improved crops, pest-resistant plants, and a cheaper source of important proteins or drugs. The social relevance of genetic engineering received official sanction in 1980 when the U.S. Supreme Court ruled that genetically modified (...) organisms can be patented. In this article I have tried to describe the discovery of the DNA:RNA hybridization technique as the successful outcome of years of intelligent and patient research in many laboratories, of inductive and deductive processes in the minds of many biologists. The synthesis that led to the final result and to the early development of the technique was made possible by the coming together of two brilliant scientists, Sol Spiegelman and Benjamin Hall. (shrink)

During the past few years significant progress has been made in our understanding of the structure and function of the proteins involved in eukaryotic DNA replication. Data from several laboratories suggest that, in contrast to prokaryotic DNA replication, two distinct DNA polymerases are required for eukaryotic DNA replication, i.e. DNA polymerase delta for the synthesis of the leading strand and DNA polymerase alpha for the lagging strand. Several accessory proteins analogous to prokaryotic replication factors have been identified and some (...) of these are specific for pol delta whereas others affect both DNA replicases. The replicases and their accessory proteins appear to be highly conserved in eukaryotes, as homologous proteins have been found in species ranging from humans to yeast. (shrink)

The ethically controversial option of genetic population screening used to be restricted to a small number of rather rare diseases by methodological limitations which are now about to be overcome. With the new technology of DNA microarrays ("DNA chip"), emerging from the synthesis of microelectronics and molecular biology, methods are now at hand for the development of mass screening programmes for a wide spectrum of genetic traits. Thus, the DNA chip may be the key technology for a refined preventive (...) medicine as well as a new dimension of eugenics. The forthcoming introduction of the DNA chip technology into medical practice urgently requires an internationally consistent framework of ethical standards and legal limitations if we do not want it to become a new Pandora's box. (shrink)

Mutations in specific genes result in birth defects, cancer, inherited diseases or lethality. The frequency with which DNA damage is converted to mutations increases dramatically when the cellular genome is replicated. Although DNA damage poses special problems to the fidelity of DNA replication, efficient mechanisms exist in mammalian cells which function to replicate their genome despite the presence of many damaged sites. These mechanisms operate in either error‐prone or error‐free modes of DNA synthesis, and frequently involve DNA strand‐pairing reactions. (...) Genetic studies in yeast and other eukaryotes suggest that replication through DNA damage is highly regulated and catalysed by complex biochemical machineries composed of many specialised gene products. Knowledge of the molecular details by which such factors facilitate the replication of damaged DNA in mammalian cells should reveal basic rules about how DNA damage induces mutagenesis and carcinogenesis. (shrink)

The postulate that a stalled/collapsed replication fork will be generated when the replication complex encounters a UV‐induced lesion in the template for leading‐strand DNA synthesis is based on the model of semi‐discontinuous DNA replication. A review of existing data indicates that the semi‐discontinuous DNA replication model is supported by data from in vitro studies, while the discontinuous DNA replication model is supported by in vivo studies in Escherichia coli. Until the question of whether DNA replicates discontinuously in one or (...) both strands is clearly resolved, any model building based on either one of the two DNA replication models should be treated with caution. BioEssays 27:633–636, 2005. © 2005 Wiley Periodicals, Inc. (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)

The RAD6 pathway of budding yeast, Saccharomyces cerevisiae, is responsible for a substantial fraction of this organism's resistance to DNA damage, and also for induced mutagenesis. The pathway appears to incorporate two different recovery processes, both regulated by RAD6. The error‐prone recovery prcess accounts for only a small amount of RAD6‐dependent resistance, but probably all induced mutagenesis. The underlying mechanism, for error‐prone recovery is very likely to be translesion synthesis. The error‐free recovery process accounts for most of RAD6‐dependent resistace, (...) but its mechanism is less clear; it may entail error‐free bypass by template switching and/or DNA gap filling by recombination. RAD6 regulates these activities by ubiquitinateins, and the roles they play in error‐free and error‐prone recovery, have not yet been established. (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)

DNA double‐strand breaks (DSBs) are extremely hazardous lesions for all DNA‐bearing organisms and the mechanisms of DSB repair are highly conserved. In the eukaryotic mitotic cell cycle, DSBs are often present following DNA replication while, in meiosis, hundreds of DSBs are generated as a prelude to the reshuffling of the maternally and paternally derived genomes. In both cases, the DSBs are repaired by a process called homologous recombinational repair (HRR), which utilises an intact DNA molecule as the repair template. Mitotic (...) and meiotic HRR are managed by ‘checkpoints’ that inhibit cell division until DSB repair is complete. Here we attempt to summarise the substantial recent progress in understanding the checkpoint management of HRR in mitosis (focussing mainly on mammals) and then go on to use this information as a framework for understanding the presumed checkpoint management of HRR in mammalian meiosis. BioEssays 29:974–986, 2007. © 2007 Wiley Periodicals, Inc. (shrink)

Inhibiting the progress of replication forks in E. coli makes them susceptible to breakage. Broken replication forks are evidently reassembled by the RecBCD recombinational repair pathway. These findings explain a particular pattern of DNA degradation during inhibition of chromosomal replication, the role of recombination in the viability of mutants with displaced replication origin, and hyper‐recombination observed in the Terminus of the E. coli chromosome in rnh mutants. Breakage and repair of inhibited replication forks could be the reason for the (...) recombination‐dependence of inducible stable DNA replication. A mechanism by which RecABCD‐dependent recombination between very short inverted repeats may help E. coli to invert an operon, transcribed in the direction opposite to that of DNA replication, is discussed. (shrink)

RNA polymerase II is recruited to DNA double‐strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage‐induced long non‐coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA‐like molecules or degraded by different ribonucleases. They can also form double‐stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to (...) the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR. (shrink)

Rifamycin is a clinically useful macrolide antibiotic produced by the gram positive bacterium. Amycolatopsis mediterranei. This antibiotic is primarily used against Mycobacterium tuberculosis and Mycobacterium leprae, causative agents of tuberculosis and leprosy, respectively. In these bacteria, rifamycin treatment specifically inhibits the initiation of RNA synthesis by binding to β‐subunit of RNA polymerase. Apart from its activity against the bacteria, rifamycin has also been reported to inhibit reverse transcriptase (RT) of certain RNA viruses. Recently, rifamycin derivatives have been dis‐covered that (...) are effective against Mycobacterium avium, which is associated with the AIDS complex. Consequently, the importance of and demand for rifamycin has increased tremendously, the world over. In this article, recent trends in rifamycin research and accessability of recombinant DNA techniques to increase rifamycin production are reviewed. (shrink)

Replication protein A (RPA), the major single‐stranded DNA‐binding protein in eukaryotic cells, is required for processing of single‐stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high‐affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that (...) while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error‐prone repair of double‐stranded breaks and promote error‐free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. (shrink)

Evolutionary theory assumed that mutations occur constantly, gradually, and randomly over time. This formulation from the “modern synthesis” of the 1930s was embraced decades before molecular understanding of genes or mutations. Since then, our labs and others have elucidated mutation mechanisms activated by stress responses. Stress‐induced mutation mechanisms produce mutations, potentially accelerating evolution, specifically when cells are maladapted to their environment, that is, when they are stressed. The mechanisms of stress‐induced mutation that are being revealed experimentally in laboratory settings (...) provide compelling models for mutagenesis that propels pathogen–host adaptation, antibiotic resistance, cancer progression and resistance, and perhaps much of evolution generally. We discuss double‐strand‐break‐dependent stress‐induced mutation in Escherichia coli. Recent results illustrate how a stress response activates mutagenesis and demonstrate this mechanism's generality and importance to spontaneous mutation. New data also suggest a possible harmony between previous, apparently opposed, models for the molecular mechanism. They additionally strengthen the case for anti‐evolvability therapeutics for infectious disease and cancer. (shrink)

The concept of regulatory ‘checkpoints’ in the eukaryotic cycle has proved to be a fruitful one. Here, its applicability to the bacterial cell cycle is examined. A primitive DNA damage checkpoint operates in E. coli such that, after exposure to ultraviolet light, while excision repair occurs, chromosome replication continues very slowly with the production of discontinuous daughter strands. The slower the rate of excision of photoproducts, the greater the delay before the normal rate of DNA replication is restored, the additional (...) time for repair ensuring that normal survival is maintained. A model is proposed in which replication rate is controlled by the ratio of RecAcoated to uncoated single stranded regions of DNA in the replication fork. There are also two cell division inhibitors SulA (=SfiA) and SfiC under the control of the SOS system and sensitive to DNA damage, but they are irrelevant to the survival of wild‐type bacteria under normal conditions. In strains where SulA and SfiC do not operate, inhibition is not influenced by the rate of excision repair and so fails one of the criteria for a DNA damage checkpoint, namely the monitoring of the DNA for the level of residual damage. (shrink)

The astonishing efficiency and accuracy of DNA replication has long suggested that refined rules enforce a single highly reproducible sequence of molecular events during the process. This view was solidified by early demonstrations that DNA unwinding and synthesis are coupled within a stable molecular factory, known as the replisome, which consists of conserved components that each play unique and complementary roles. However, recent single-molecule observations of replisome dynamics have begun to challenge this view, revealing that replication may not be (...) defined by a uniform sequence of events. Instead, multiple exchange pathways, pauses, and DNA loop types appear to dominate replisome function. These observations suggest we must rethink our fundamental assumptions and acknowledge that each replication cycle may involve sampling of alternative, sometimes parallel, pathways. Here, we review our current mechanistic understanding of DNA replication while highlighting findings that exemplify multi-pathway aspects of replisome function and considering the broader implications. Recent single-molecule studies have revealed that replisome function is dominated by stochastic sampling of multiple molecular pathways. In the context of these new findings, we re-examine the foundational assumptions and seminal experiments that have guided our understanding of replication mechanism and reconcile several long-debated coordination models. (shrink)

In many mammalian cell types, increases in the level of nonpolymerized tubulin cause an inhibition in tubulin synthesis which is accompanied by a decrease in tubulin mRNA levels. To see whether inhibition is caused by nuclear or cytoplasmic events, two groups have recently examined the ability of enucleated cells to autoregulate tubulin synthesis.1,2 These experiments have demonstrated that transcription, processing, and transport of tubulin mRNAs from the nucleus to the cytoplasm are not major sites of autoregulation. (...) Instead, monomeric tubulin must reduce, either directly or indirectly, the translatability of its own message. (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)

DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases δ and ε. It now appears that proliferating cell (...) nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post‐replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells. (shrink)

The Modern Synthesis has dominated biology for 80 years. It was formulated in 1942, a decade before the major achievements of molecular biology, including the Double Helix and the Central Dogma. When first formulated in the 1950s these discoveries and concepts seemed initially to completely justify the central genetic assumptions of the Modern Synthesis. The Double Helix provided the basis for highly accurate DNA replication, while the Central Dogma was viewed as supporting the Weismann Barrier, so excluding the (...) inheritance of acquired characteristics. This article examines the language of the Modern Synthesis and reveals that it is based on four important misinterpretations of what molecular biology had shown, so forming the basis of the four Illusions: 1. Natural Selection; 2. The Weismann Barrier; 3. The Rejection of Darwin’s Gemmules; 4. The Central Dogma. A multi-level organisation view of biology avoids these illusions through the principle of biological relativity. Molecular biology does not therefore confirm the assumptions of the Modern Synthesis. (shrink)

Chromosomal origins of DNA replication in higher eukaryotes differ significantly from those of E. coli (oriC) and the tumor virus, SV40 (ori sequence). Initiation events appear to occur throughout broad zones rather than at specific origin sequences. Analysis of four chromosomal origin regions reveals that they share common modular sequence elements. These include DNA unwinding elements, pyrimidine tracts that may serve as strong DNA polymerase‐primase start sites, scaffold associated regions, transcriptional regulatory sequences, and, possibly, initiator protein binding sites and inherently (...) destabilized regions. Based on the novel organization of chromosomal origin regions, we propose a model for initiation of DNA replication in higher eukaryotes. Unwinding of duplex DNA during initiation may be uncoupled, both temporally and spatially, from DNA synthesis, resulting in transient single‐stranded intermediates that function in lieu of conventional replication forks during chromosomal DNA replication. DNA synthesis begins subsequently at multiple sites Within the unwound regions rather than at specific origin sequences. (shrink)