Elevated MSH2 MSH3 expression interferes with DNA metabolism in vivo.

The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.

Preserved appendages in a Silurian binodicope: implications for the evolutionary history of ostracod crustaceans.

Ostracod crustaceans originated at least 500 Ma ago. Their tiny bivalved shells represent the most species-abundant fossil arthropods, and ostracods are omnipresent in a wide array of freshwater and marine environments today and in the past. Derima paparme gen. et sp. nov. from the Herefordshire Silurian Lagerstätte (~430 Ma) in the Welsh Borderland, UK, is one of only a handful of exceptionally preserved ostracods (with soft parts as well as the shell) known from the Palaeozoic. A male specimen provides the first evidence of the appendages of Binodicopina, a major group of Palaeozoic ostracods comprising some 135 Ordovician to Permian genera. The appendage morphology of D. paparme, but not its shell, indicates that binodicopes belong to Podocopa. The discovery that the soft-part morphology of binodicopes allies them with podocopes affirms that using the shell alone is an unreliable basis for classifying certain fossil ostracods, and knowledge of soft-part morphology is critical for the task. Current assignment of many fossil ostracods to higher taxa, and therefore the evolutionary history of the group, may require reconsideration.

During Translesion Synthesis, Escherichia coli DinB89 (T120P) Alters Interactions of DinB (Pol IV) with Pol III Subunit Assemblies and SSB, but Not with the ß Clamp.

Translesion synthesis (TLS) by specialized DNA polymerases (Pols) is an evolutionarily conserved mechanism for tolerating replication-blocking DNA lesions. Using the Escherichia coli dinB-encoded Pol IV as a model to understand how TLS is coordinated with the actions of the high-fidelity Pol III replicase, we previously described a novel Pol IV mutant containing a threonine 120-to-proline mutation (Pol IV-T120P) that failed to exchange places with Pol III at the replication fork in vitro as part of a Pol III-Pol IV switch. This in vitro defect correlated with the inability of Pol IV-T120P to support TLS in vivo, suggesting Pol IV gains access to the DNA, at least in part, via a Pol III-Pol IV switch. Interaction of Pol IV with the ß sliding clamp and the single-stranded DNA binding protein (SSB) significantly stimulates Pol IV replication and facilitates its access to the DNA. In this work, we demonstrate that Pol IV interacts physically with Pol III. We further show that Pol IV-T120P interacts normally with the ß clamp, but is impaired in interactions with the α catalytic and εθ proofreading subunits of Pol III, as well as SSB. Taken together with published work, these results provide strong support for the model in which Pol IV-Pol III and Pol IV-SSB interactions help to regulate the access of Pol IV to the DNA. Finally, we describe several additional E. coli Pol-Pol interactions, suggesting Pol-Pol interactions play fundamental roles in coordinating bacterial DNA replication, DNA repair, and TLS. IMPORTANCE Specialized DNA polymerases (Pols) capable of catalyzing translesion synthesis (TLS) generate mutations that contribute to bacterial virulence, pathoadaptation, and antimicrobial resistance. One mechanism by which the bacterial TLS Pol IV gains access to the DNA to generate mutations is by exchanging places with the bacterial Pol III replicase via a Pol III-Pol IV switch. Here, we describe multiple Pol III-Pol IV interactions and discuss evidence that these interactions are required for the Pol III-Pol IV switch. Furthermore, we describe several additional E. coli Pol-Pol interactions that may play fundamental roles in managing the actions of the different bacterial Pols in DNA replication, DNA repair, and TLS.

Elevated Levels of the Escherichia coli nrdAB-Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the ß Clamp.

Expression of the Escherichia coli dnaN-encoded ß clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (ßE202K bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaNE202K strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB, that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the ß clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaNE202K allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the ßE202K and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB-encoded RNR relieved the growth defect caused by elevated levels of the ß clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated ß clamp levels to impede growth and suggest either that multiple effects stemming from the dnaNE202K mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the ß clamp-Hda complex. The dnaNE202K allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaNE202K strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and ßE202K clamp proteins support equivalent levels of Hda activity in vitro. Taken together, these results suggest that ßE202K-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.

The Mutant ßE202K Sliding Clamp Protein Impairs DNA Polymerase III Replication Activity.

Expression of the Escherichia coli dnaN-encoded ß clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess ß clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro. Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the ßE202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still sequester Pol III effectively. Of interest, ßE202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaNE202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant ßE202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the ß clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The ß clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the ß clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant ß clamps that fail to inhibit growth. Their analysis revealed that ßE202K is unique among them. Our work offers new insights into how the ß clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV.

Role of the SOS Response in the Generation of Antibiotic Resistance In Vivo.

The SOS response to DNA damage is a conserved stress response in Gram-negative and Gram-positive bacteria. Although this pathway has been studied for years, its relevance is still not familiar to many working in the fields of clinical antibiotic resistance and stewardship. Under some conditions, the SOS response favors DNA repair and preserves the genetic integrity of the organism. On the other hand, the SOS response also includes induction of error-prone DNA polymerases, which can increase the rate of mutation, called the mutator phenotype or "hypermutation." As a result, mutations can occur in genes conferring antibiotic resistance, increasing the acquisition of resistance to antibiotics. Almost all of the work on the SOS response has been on bacteria exposed to stressors in vitro. In this study, we sought to quantitate the effects of SOS-inducing drugs in vivo, in comparison with the same drugs in vitro. We used a rabbit model of intestinal infection with enteropathogenic Escherichia coli strain E22. SOS-inducing drugs triggered the mutator phenotype response in vivo as well as in vitro. Exposure of E. coli strain E22 to ciprofloxacin or zidovudine, both of which induce the SOS response in vitro, resulted in increased antibiotic resistance to 3 antibiotics: rifampin, minocycline, and fosfomycin. Zinc was able to inhibit the SOS-induced emergence of antibiotic resistance in vivo, as previously observed in vitro. Our findings may have relevance in reducing the emergence of resistance to new antimicrobial drugs.

Evolutionary simulations clarify and reconcile biodiversity-disturbance models.

There is significant geographic variation in species richness. However, the nature of the underlying relationships, such as that between species richness and environmental stability, remains unclear. The stability-time hypothesis suggests that environmental instability reduces species richness by suppressing speciation and increasing extinction risk. By contrast, the patch-mosaic hypothesis suggests that small-scale environmental instability can increase species richness by providing a steady supply of non-equilibrium environments. Although these hypotheses are often applied to different time scales, their core mechanisms are in conflict. Reconciling these apparently competing hypotheses is key to understanding how environmental conditions shape the distribution of biodiversity. Here, we use REvoSim, an individual-based, eco-evolutionary system, to model the evolution of sessile organisms in environments with varying magnitudes and scales of environmental instability. We demonstrate that when environments have substantial permanent heterogeneity, a high level of localized environmental instability reduces biodiversity, whereas in environments lacking permanent heterogeneity, high levels of localized instability increase biodiversity. By contrast, broad-scale environmental instability, acting on the same time scale, invariably reduces biodiversity. Our results provide a new view of the biodiversity-disturbance relationship that reconciles contrasting hypotheses within a single model and implies constraints on the environmental conditions under which those hypotheses apply. These constraints can inform attempts to conserve adaptive potential in different environments during the current biodiversity crisis.

Morphological Phylogenetics Evaluated Using Novel Evolutionary Simulations.

Evolutionary inferences require reliable phylogenies. Morphological data have traditionally been analyzed using maximum parsimony, but recent simulation studies have suggested that Bayesian analyses yield more accurate trees. This debate is ongoing, in part, because of ambiguity over modes of morphological evolution and a lack of appropriate models. Here, we investigate phylogenetic methods using two novel simulation models-one in which morphological characters evolve stochastically along lineages and another in which individuals undergo selection. Both models generate character data and lineage splitting simultaneously: the resulting trees are an emergent property, rather than a fixed parameter. Standard consensus methods for Bayesian searches (Mki) yield fewer incorrect nodes and quartets than the standard consensus trees recovered using equal weighting and implied weighting parsimony searches. Distances between the pool of derived trees (most parsimonious or posterior distribution) and the true trees-measured using Robinson-Foulds (RF), subtree prune and regraft (SPR), and tree bisection reconnection (TBR) metrics-demonstrate that this is related to the search strategy and consensus method of each technique. The amount and structure of homoplasy in character data differ between models. Morphological coherence, which has previously not been considered in this context, proves to be a more important factor for phylogenetic accuracy than homoplasy. Selection-based models exhibit relatively lower homoplasy, lower morphological coherence, and higher inaccuracy in inferred trees. Selection is a dominant driver of morphological evolution, but we demonstrate that it has a confounding effect on numerous character properties which are fundamental to phylogenetic inference. We suggest that the current debate should move beyond considerations of parsimony versus Bayesian, toward identifying modes of morphological evolution and using these to build models for probabilistic search methods. [Bayesian; evolution; morphology; parsimony; phylogenetics; selection; simulation.].

Binding of the regulatory domain of MutL to the sliding ß-clamp is species specific.

The ß-clamp is a protein hub central to DNA replication and fork management. Proteins interacting with the ß-clamp harbor a conserved clamp-binding motif that is often found in extended regions. Therefore, clamp interactions have -almost exclusively- been studied using short peptides recapitulating the binding motif. This approach has revealed the molecular determinants that mediate the binding but cannot describe how proteins with clamp-binding motifs embedded in structured domains are recognized. The mismatch repair protein MutL has an internal clamp-binding motif, but its interaction with the ß-clamp has different roles depending on the organism. In Bacillus subtilis, the interaction stimulates the endonuclease activity of MutL and it is critical for DNA mismatch repair. Conversely, disrupting the interaction between Escherichia coli MutL and the ß-clamp only causes a mild mutator phenotype. Here, we determined the structures of the regulatory domains of E. coli and B. subtilis MutL bound to their respective ß-clamps. The structures reveal different binding modes consistent with the binding to the ß-clamp being a two-step process. Functional characterization indicates that, within the regulatory domain, only the clamp binding motif is required for the interaction between the two proteins. However, additional motifs beyond the regulatory domain may stabilize the interaction. We propose a model for the activation of the endonuclease activity of MutL in organisms lacking methyl-directed mismatch repair.

A new ophiocistioid with soft-tissue preservation from the Silurian Herefordshire Lagerstätte, and the evolution of the holothurian body plan.

Reconstructing the evolutionary assembly of animal body plans is challenging when there are large morphological gaps between extant sister taxa, as in the case of echinozoans (echinoids and holothurians). However, the inclusion of extinct taxa can help bridge these gaps. Here we describe a new species of echinozoan, Sollasina cthulhu, from the Silurian Herefordshire Lagerstätte, UK. Sollasina cthulhu belongs to the ophiocistioids, an extinct group that shares characters with both echinoids and holothurians. Using physical-optical tomography and computer reconstruction, we visualize the internal anatomy of S. cthulhu in three dimensions, revealing inner soft tissues that we interpret as the ring canal, a key part of the water vascular system that was previously unknown in fossil echinozoans. Phylogenetic analyses strongly suggest that Sollasina and other ophiocistioids represent a paraphyletic group of stem holothurians, as previously hypothesized. This allows us to reconstruct the stepwise reduction of the skeleton during the assembly of the holothurian body plan, which may have been controlled by changes in the expression of biomineralization genes.

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing.

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-ß clamp complex. This complex contains two pairs of Hda dimers sandwiched between two ß clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the ß clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-ß clamp complex indicate that the interaction of the ß clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-ß clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function.

Tiny individuals attached to a new Silurian arthropod suggest a unique mode of brood care.

The ∼430-My-old Herefordshire, United Kingdom, Lagerstätte has yielded a diversity of remarkably preserved invertebrates, many of which provide fundamental insights into the evolutionary history and ecology of particular taxa. Here we report a new arthropod with 10 tiny arthropods tethered to its tergites by long individual threads. The head of the host, which is covered by a shield that projects anteriorly, bears a long stout uniramous antenna and a chelate limb followed by two biramous appendages. The trunk comprises 11 segments, all bearing limbs and covered by tergites with long slender lateral spines. A short telson bears long parallel cerci. Our phylogenetic analysis resolves the new arthropod as a stem-group mandibulate. The evidence suggests that the tethered individuals are juveniles and the association represents a complex brooding behavior. Alternative possibilities-that the tethered individuals represent a different epizoic or parasitic arthropod-appear less likely.

Insufficient levels of the nrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. coli strain.

The ATP-bound form of the Escherichia coli DnaA replication initiator protein remodels the chromosomal origin of replication, oriC, to load the replicative helicase. The primary mechanism for regulating the activity of DnaA involves the Hda and ß clamp proteins, which act together to dramatically stimulate the intrinsic DNA-dependent ATPase activity of DnaA via a process termed Regulatory Inactivation of DnaA. In addition to hyperinitiation, strains lacking hda function also exhibit cold sensitive growth at 30°C. Strains impaired for the other regulators of initiation (i.e., ΔseqA or ΔdatA) fail to exhibit cold sensitivity. The goal of this study was to gain insight into why loss of hda function impedes growth. We used a genetic approach to isolate 9 suppressors of Δhda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δhda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δhda is diminished levels of DNA precursors, particularly dGTP and dATP. We discuss possible mechanisms by which the suppressors identified here may regulate dNTP pool size, as well as similarities in phenotypes between the Δhda strain and hda+ strains exposed to the ribonucleotide reductase inhibitor hydroxyurea.

A well-preserved respiratory system in a Silurian ostracod.

Ostracod crustaceans are diverse and ubiquitous in aqueous environments today but relatively few known species have gills. Ostracods are the most abundant fossil arthropods but examples of soft-part preservation, especially of gills, are exceptionally rare. A new ostracod, Spiricopia aurita (Myodocopa), from the marine Silurian Herefordshire Lagerstätte (430 Mya), UK, preserves appendages, lateral eyes and gills. The respiratory system includes five pairs of gill lamellae with hypobranchial and epibranchial canals that conveyed haemolymph. A heart and associated vessels had likely evolved in ostracods by the Mid-Silurian.

A Silurian armoured aplacophoran and implications for molluscan phylogeny.

The Mollusca is one of the most diverse, important and well-studied invertebrate phyla; however, relationships among major molluscan taxa have long been a subject of controversy. In particular, the position of the shell-less vermiform Aplacophora and its relationship to the better-known Polyplacophora (chitons) have been problematic: Aplacophora has been treated as a paraphyletic or monophyletic group at the base of the Mollusca, proximate to other derived clades such as Cephalopoda, or as sister group to the Polyplacophora, forming the clade Aculifera. Resolution of this debate is required to allow the evolutionary origins of Mollusca to be reconstructed with confidence. Recent fossil finds support the Aculifera hypothesis, demonstrating that the Palaeozoic-era palaeoloricate 'chitons' included taxa combining certain polyplacophoran and aplacophoran characteristics. However, fossils combining an unambiguously aplacophoran-like body with chiton-like valves have remained elusive. Here we describe such a fossil, Kulindroplax perissokomos gen. et sp. nov., from the Herefordshire Lagerstätte (about 425 million years bp), a Silurian deposit preserving a marine biota in unusual three-dimensional detail. The specimen is reconstructed three-dimensionally through physical-optical tomography. Phylogenetic analysis indicates that this and many other palaeoloricate chitons are crown-group aplacophorans.

Exchange between Escherichia coli polymerases II and III on a processivity clamp.

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, ß. Single-molecule experiments reveal that the interactions of Pol II and Pol III with ß allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a ß-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.

A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis.

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the ß sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the ß clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.

DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks.

Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III α that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a τ-containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ showed that τ-, τ2-, or τ3-DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.

An edrioasteroid from the Silurian Herefordshire Lagerstätte of England reveals the nature of the water vascular system in an extinct echinoderm.

Echinoderms are unique in having a water vascular system with tube feet, which perform a variety of functions in living forms. Here, we report the first example of preserved tube feet in an extinct group of echinoderms. The material, from the Silurian Herefordshire Lagerstätte, UK, is assigned to a new genus and species of rhenopyrgid edrioasteroid, Heropyrgus disterminus The tube feet attach to the inner surface of compound interradial plates and form two sets, an upper and a lower, an arrangement never reported previously in an extant or extinct echinoderm. Cover plates are absent and floor plates are separated creating a large permanent entrance to the interior of the oral area. The tube feet may have captured food particles that entered the oral area and/or enhanced respiration. The pentameral symmetry of the oral surface transitions to eight columns in which the plates are vertically offset resulting in a spiral appearance. This change in symmetry may reflect flexibility in the evolutionary development of the axial and extraxial zones in early echinoderm evolution.

A new crustacean from the Herefordshire (Silurian) Lagerstätte, UK, and its significance in malacostracan evolution.

Cascolus ravitis gen. et sp. nov. is a three-dimensionally preserved fossil crustacean with soft parts from the Herefordshire (Silurian) Lagerstätte, UK. It is characterized by a head with a head shield and five limb pairs, and a thorax (pereon) with nine appendage-bearing segments followed by an apodous abdomen (pleon). All the appendages except the first are biramous and have a gnathobase. The post-mandibular appendages are similar one to another, and bear petal-shaped epipods that probably functioned as a part of the respiratory-circulatory system. Cladistic analysis resolves the new taxon as a stem-group leptostracan (Malacostraca). This well-preserved arthropod provides novel insights into the evolution of appendage morphology, tagmosis and the possible respiratory-circulatory physiology of a basal malacostracan.