Physical and Functional Compartmentalization of Archaeal Chromosomes.

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."

Multi-scale architecture of archaeal chromosomes.

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to providing high-resolution descriptions of archaeal chromosome architectures, our data provide evidence of multiple modes of organization in prokaryotic chromosomes and yield insights into the evolution of eukaryotic chromosome conformation.

Capturing chromosome conformation in Crenarchaea.

While there is a considerable body of knowledge regarding the molecular and structural biology and biochemistry of archaeal information processing machineries, far less is known about the nature of the substrate for these machineries-the archaeal nucleoid. In this article, we will describe recent advances in our understanding of the three-dimensional organization of the chromosomes of model organisms in the crenarchaeal phylum.

Archaeal DNA Replication.

It is now well recognized that the information processing machineries of archaea are far more closely related to those of eukaryotes than to those of their prokaryotic cousins, the bacteria. Extensive studies have been performed on the structure and function of the archaeal DNA replication origins, the proteins that define them, and the macromolecular assemblies that drive DNA unwinding and nascent strand synthesis. The results from various archaeal organisms across the archaeal domain of life show surprising levels of diversity at many levels-ranging from cell cycle organization to chromosome ploidy to replication mode and nature of the replicative polymerases. In the following, we describe recent advances in the field, highlighting conserved features and lineage-specific innovations.

Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins.

Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins.

Emerging views of genome organization in Archaea.

Over the past decade, advances in methodologies for the determination of chromosome conformation have provided remarkable insight into the local and higher-order organization of bacterial and eukaryotic chromosomes. Locally folded domains are found in both bacterial and eukaryotic genomes, although they vary in size. Importantly, genomes of metazoans also possess higher-order organization into A- and B-type compartments, regions of transcriptionally active and inactive chromatin, respectively. Until recently, nothing was known about the organization of genomes of organisms in the third domain of life - the archaea. However, despite archaea possessing simple circular genomes that are morphologically reminiscent of those seen in many bacteria, a recent study of archaea of the genus Sulfolobus has revealed that it organizes its genome into large-scale domains. These domains further interact to form defined A- and B-type compartments. The interplay of transcription and localization of a novel structural maintenance of chromosomes (SMC) superfamily protein, termed coalescin, defines compartment identity. In this Review, we discuss the mechanistic and evolutionary implications of these findings.

Form and function of archaeal genomes.

A key maxim in modernist architecture is that 'form follows function'. While modernist buildings are hopefully the product of intelligent design, the architectures of chromosomes have been sculpted by the forces of evolution over many thousands of generations. In the following, I will describe recent advances in our understanding of chromosome architecture in the archaeal domain of life. Although much remains to be learned about the mechanistic details of archaeal chromosome organization, some general principles have emerged. At the 10-100 kb level, archaeal chromosomes have a conserved local organization reminiscent of bacterial genomes. In contrast, lineage-specific innovations appear to have imposed distinct large-scale architectural features. The ultimate functions of genomes are to store and to express genetic information. Gene expression profiles have been shown to influence chromosome architecture, thus their form follows function. However, local changes to chromosome conformation can also influence gene expression and therefore, in these instances, function follows form.

MCM loading--an open-and-shut case?

Two recent studies, from Fernández-Cid et al. (2013) (this issue of Molecular Cell) and Frigola et al. (2013), have elegantly dissected key events and interactions in the loading of the budding yeast replicative helicase MCM(2-7).

An archaeal primase functions as a nanoscale caliper to define primer length.

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3' OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper.

Extra-chromosomal elements and the evolution of cellular DNA replication machineries.

DNA replication is fundamental to the propagation of cellular life. Remarkably, the bacterial replication machinery is distinct from that used by archaea and eukaryotes. In this article, we discuss the role that lateral gene transfer by extra-chromosomal elements might have had in shaping the replication machinery and even modulating the manner in which host cellular genomes are replicated.

Initiating DNA replication: a matter of prime importance.

It has been known for decades that the principal replicative DNA polymerases that effect genome replication are incapable of starting DNA synthesis de novo Rather, they require a 3'-OH group from which to extend a DNA chain. Cellular DNA replication systems exploit a dedicated, limited processivity RNA polymerase, termed primase, that synthesizes a short oligoribonucleotide primer which is then extended by a DNA polymerase. Thus, primases can initiate synthesis, proceed with primer elongation for a short distance then transfer the primer to a DNA polymerase. Despite these well-established properties, the mechanistic basis of these dynamic behaviours has only recently been established. In the following, the author will describe recent insights from studies of the related eukaryotic and archaeal DNA primases. Significantly, the general conclusions from these studies likely extend to a broad class of extrachromosomal element-associated primases as well as the human primase-related DNA repair enzyme, PrimPol.

Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division.

Members of the crenarchaeal kingdom, such as Sulfolobus, divide by binary fission yet lack genes for the otherwise near-ubiquitous tubulin and actin superfamilies of cytoskeletal proteins. Recent work has established that Sulfolobus homologs of the eukaryotic ESCRT-III and Vps4 components of the ESCRT machinery play an important role in Sulfolobus cell division. In eukaryotes, several pathways recruit ESCRT-III proteins to their sites of action. However, the positioning determinants for archaeal ESCRT-III are not known. Here, we identify a protein, CdvA, that is responsible for recruiting Sulfolobus ESCRT-III to membranes. Overexpression of the isolated ESCRT-III domain that interacts with CdvA results in the generation of nucleoid-free cells. Furthermore, CdvA and ESCRT-III synergize to deform archaeal membranes in vitro. The structure of the CdvA/ESCRT-III interface gives insight into the evolution of the more complex and modular eukaryotic ESCRT complex.

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM.

The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS•Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome.

Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends.

Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional 'brush-like' structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms.

The Structure, Function and Roles of the Archaeal ESCRT Apparatus.

Although morphologically resembling bacteria, archaea constitute a distinct domain of life with a closer affiliation to eukaryotes than to bacteria. This similarity is seen in the machineries for a number of essential cellular processes, including DNA replication and gene transcription. Perhaps surprisingly, given their prokaryotic morphology, some archaea also possess a core cell division apparatus that is related to that involved in the final stages of membrane abscission in vertebrate cells, the ESCRT machinery.

Initiation of DNA Replication in the Archaea.

Organisms within the archaeal domain of life possess a simplified version of the eukaryotic DNA replication machinery. While some archaea possess a bacterial-like mode of DNA replication with single origins of replication per chromosome, the majority of species characterized to date possess chromosomes with multiple replication origins. Genetic, structural, and biochemical studies have revealed the nature of archaeal origin specification. Recent work has begun to shed light on the mechanisms of replication initiation in these organisms.

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation.

Chromosomal DNA replication requires one daughter strand-the lagging strand-to be synthesised as a series of discontinuous, RNA-primed Okazaki fragments, which must subsequently be matured into a single covalent DNA strand. Here, we describe the reconstitution of Okazaki fragment maturation in vitro using proteins derived from the archaeon Sulfolobus solfataricus. Six proteins are necessary and sufficient for coupled DNA synthesis, RNA primer removal and DNA ligation. PolB1, Fen1 and Lig1 provide the required catalytic activities, with coordination of their activities dependent upon the DNA sliding clamp, proliferating cell nuclear antigen (PCNA). S. solfataricus PCNA is a heterotrimer, with each subunit having a distinct specificity for binding PolB1, Fen1 or Lig1. Our data demonstrate that the most efficient coupling of activities occurs when a single PCNA ring organises PolB1, Fen1 and Lig1 into a complex.

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus.

DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNA-polymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins.

Calorie restriction does not restore brain mitochondrial function in P301L tau mice, but it does decrease mitochondrial F0F1-ATPase activity.

Calorie restriction (CR) has been shown to increase lifespan and delay aging phenotypes in many diverse eukaryotic species. In mouse models of Alzheimer's disease (AD), CR has been shown to decrease amyloid-beta and hyperphosphorylated tau levels and preserve cognitive function. Overexpression of human mutant tau protein has been shown to induce deficits in mitochondrial electron transport chain complex I activity. Therefore, experiments were performed to determine the effects of 4-month CR on brain mitochondrial function in Tg4510 mice, which express human P301L tau. Expression of mutant tau led to decreased ADP-stimulated respiratory rates, but not uncoupler-stimulated respiratory rates. The membrane potential was also slightly higher in mitochondria from the P301L tau mice. As shown previously, tau expression decreased mitochondrial complex I activity. The decreased complex I activity, decreased ADP-stimulated respiratory rate, and increased mitochondrial membrane potential occurring in mitochondria from Tg4510 mice were not restored by CR. However, the CR diet did result in a genotype independent decrease in mitochondrial F0F1-ATPase activity. This decrease in F0F1-ATPase activity was not due to lowered levels of the alpha or beta subunits of F0F1-ATPase. The possible mechanisms through which CR reduces the F0F1-ATPase activity in brain mitochondria are discussed.

Functional interplay between a virus and the ESCRT machinery in archaea.

Recently it has been discovered that a number of eukaryotic viruses, including HIV, coopt the cellular Endosomal Sorting Complex Required for Transport (ESCRT) machinery to affect egress from infected cells. Strikingly, the ESCRT apparatus is conserved in a subset of Archaea, including members of the genus Sulfolobus where it plays a role in cytokinesis. In the current work, we reveal that the archaeal virus Sulfolobus turreted icosahedral virus isolated from Yellowstone National Park's acidic hot springs also exploits the host ESCRT machinery in its replication cycle. Moreover, perturbation of normal ESCRT function abrogates viral replication and, thus, prevents establishment of a productive Sulfolobus turreted icosahedral virus infection. We propose that the Sulfolobus ESCRT machinery is involved in viral assembly within the cytoplasm and in escape from the infected cell by using a unique lysis mechanism. Our results support an ancient origin for viruses "hijacking" ESCRT proteins to complete their replication cycle and thus identify a critical host-virus interaction conserved between two domains of life.