The combined DNA and RNA synthetic capabilities of archaeal DNA primase facilitate primer hand-off to the replicative DNA polymerase.

Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5'-RNA-DNA-3' hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide stochastically during elongation and that this switches the primase into a dedicated DNA synthetic mode polymerase.

The regulation of DNA supercoiling across evolution.

DNA supercoiling controls a variety of cellular processes, including transcription, recombination, chromosome replication, and segregation, across all domains of life. As a physical property, DNA supercoiling alters the double helix structure by under- or over-winding it. Intriguingly, the evolution of DNA supercoiling reveals both similarities and differences in its properties and regulation across the three domains of life. Whereas all organisms exhibit local, constrained DNA supercoiling, only bacteria and archaea exhibit unconstrained global supercoiling. DNA supercoiling emerges naturally from certain cellular processes and can also be changed by enzymes called topoisomerases. While structurally and mechanistically distinct, topoisomerases that dissipate excessive supercoils exist in all domains of life. By contrast, topoisomerases that introduce positive or negative supercoils exist only in bacteria and archaea. The abundance of topoisomerases is also transcriptionally and post-transcriptionally regulated in domain-specific ways. Nucleoid-associated proteins, metabolites, and physicochemical factors influence DNA supercoiling by acting on the DNA itself or by impacting the activity of topoisomerases. Overall, the unique strategies that organisms have evolved to regulate DNA supercoiling hold significant therapeutic potential, such as bactericidal agents that target bacteria-specific processes or anticancer drugs that hinder abnormal DNA replication by acting on eukaryotic topoisomerases specialized in this process. The investigation of DNA supercoiling therefore reveals general principles, conserved mechanisms, and kingdom-specific variations relevant to a wide range of biological questions.

Archaeal DNA Replication Origins and Recruitment of the MCM Replicative Helicase.

DNA replication is fundamental to the propagation of all life on the planet. Remarkably, given the central importance for this process, two distinct core cellular DNA replication machineries have evolved. One is found in the bacterial domain of life and the other is present in Archaea and Eukarya. The archaeal machinery represents a simplified and presumably ancestral form of the eukaryotic DNA replication apparatus. As such, archaeal replication proteins have been studied extensively as models for their eukaryal counterparts. In addition, a number of archaea have been developed as model organisms. Accordingly, there has been a considerable increase in our knowledge of how archaeal chromosomes are replicated. It has become apparent that the majority of archaeal cells replicate their genomes from multiple origins per chromosome. Thus, at both organizational and mechanistic levels, archaeal DNA replication resembles that of eukarya. In this chapter, we will describe recent advances in our understanding of the basis of archaeal origin definition and how the archaeal initiator proteins recruit the replicative helicase to origins.

The roles of family B and D DNA polymerases in Thermococcus species 9°N Okazaki fragment maturation.

During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed.

Archaeal replicative primases can perform translesion DNA synthesis.

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2'-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.

O6-alkylguanine postlesion DNA synthesis is correct with the right complement of hydrogen bonding.

The ability of a DNA polymerase to replicate DNA beyond a mismatch containing a DNA lesion during postlesion DNA synthesis (PLS) can be a contributing factor to mutagenesis. In this study, we investigate the ability of Dpo4, a Y-family DNA polymerase from Sulfolobus solfataricus, to perform PLS beyond the pro-mutagenic DNA adducts O(6)-benzylguanine (O(6)-BnG) and O(6)-methylguanine (O(6)-MeG). Here, O(6)-BnG and O(6)-MeG were paired opposite artificial nucleosides that were structurally altered to systematically test the influence of hydrogen bonding and base pair size and shape on O(6)-alkylguanine PLS. Dpo4-mediated PLS was more efficient past pairs containing Benzi than pairs containing the other artificial nucleoside probes. Based on steady-state kinetic analysis, frequencies of mismatch extension were 7.4 × 10(-3) and 1.5 × 10(-3) for Benzi:O(6)-MeG and Benzi:O(6)-BnG pairs, respectively. Correct extension was observed when O(6)-BnG and O(6)-MeG were paired opposite the smaller nucleoside probes Benzi and BIM; conversely, Dpo4 did not extend past the larger nucleoside probes, Peri and Per, placed opposite O(6)-BnG and O(6)-MeG. Interestingly, Benzi was extended with high fidelity by Dpo4 when it was paired opposite O(6)-BnG and O(6)-MeG but not opposite G. These results indicate that hydrogen bonding is an important noncovalent interaction that influences the fidelity and efficiency of Dpo4 to perform high-fidelity O(6)-alkylguanine PLS.

Accelerated growth in the absence of DNA replication origins.

DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whereas most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We use deep sequencing to study replication in Haloferax volcanii and identify four chromosomal origins of differing activity. Deletion of individual origins results in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.

Cooperative dynamics of a DNA polymerase replicating complex.

Engineered DNA polymerases continue to be the workhorses of many applications in biotechnology, medicine and nanotechnology. However, the dynamic interplay between the enzyme and the DNA remains unclear. In this study, we performed an extensive replica exchange with flexible tempering (REFT) molecular dynamics simulation of the ternary replicating complex of the archaeal family B DNA polymerase from the thermophile Thermococcus gorgonarius, right before the chemical step. The convoluted dynamics of the enzyme are reducible to rigid-body motions of six subdomains. Upon binding to the enzyme, the DNA double helix conformation changes from a twisted state to a partially untwisted state. The twisted state displays strong bending motion, whereby the DNA oscillates between a straight and a bent conformation. The dynamics of double-stranded DNA are strongly correlated with rotations of the thumb toward the palm, which suggests an assisting role of the enzyme during DNA translocation. In the complex, the primer-template duplex displays increased preference for the B-DNA conformation at the n-2 and n-3 dinucleotide steps. Interactions at the primer 3' end indicate that Thr541 and Asp540 are the acceptors of the first proton transfer in the chemical step, whereas in the translocation step both residues hold the primer 3' terminus in the vicinity of the priming site, which is crucial for high processivity.

Single-molecule investigation of substrate binding kinetics and protein conformational dynamics of a B-family replicative DNA polymerase.

Replicative DNA polymerases use a complex, multistep mechanism for efficient and accurate DNA replication as uncovered by intense kinetic and structural studies. Recently, single-molecule fluorescence spectroscopy has provided new insights into real time conformational dynamics utilized by DNA polymerases during substrate binding and nucleotide incorporation. We have used single-molecule Förster resonance energy transfer techniques to investigate the kinetics and conformational dynamics of Sulfolobus solfataricus DNA polymerase B1 (PolB1) during DNA and nucleotide binding. Our experiments demonstrate that this replicative polymerase can bind to DNA in at least three conformations, corresponding to an open and closed conformation of the finger domain as well as a conformation with the DNA substrate bound to the exonuclease active site of PolB1. Additionally, our results show that PolB1 can transition between these conformations without dissociating from a primer-template DNA substrate. Furthermore, we show that the closed conformation is promoted by a matched incoming dNTP but not by a mismatched dNTP and that mismatches at the primer-template terminus lead to an increase in the binding of the DNA to the exonuclease site. Our analysis has also revealed new details of the biphasic dissociation kinetics of the polymerase-DNA binary complex. Notably, comparison of the results obtained in this study with PolB1 with those from similar single-molecule studies with an A-family DNA polymerase suggests mechanistic differences between these polymerases. In summary, our findings provide novel mechanistic insights into protein conformational dynamics and substrate binding kinetics of a high fidelity B-family DNA polymerase.

Thermococcus kodakarensis DNA replication.

DNA replication plays an essential role in all life forms. Research on archaeal DNA replication began approximately 20 years ago. Progress was hindered, however, by the lack of genetic tools to supplement the biochemical and structural studies. This has changed, however, and genetic approaches are now available for several archaeal species. One of these organisms is the thermophilic euryarchaeon Thermococcus kodakarensis. In the present paper, the recent developments in the biochemical, structural and genetic studies on the replication machinery of T. kodakarensis are summarized.

Differential temperature-dependent multimeric assemblies of replication and repair polymerases on DNA increase processivity.

Differentiation of binding accurate DNA replication polymerases over error prone DNA lesion bypass polymerases is essential for the proper maintenance of the genome. The hyperthermophilic archaeal organism Sulfolobus solfataricus (Sso) contains both a B-family replication (Dpo1) and a Y-family repair (Dpo4) polymerase and serves as a model system for understanding molecular mechanisms and assemblies for DNA replication and repair protein complexes. Protein cross-linking, isothermal titration calorimetry, and analytical ultracentrifugation have confirmed a previously unrecognized dimeric Dpo4 complex bound to DNA. Binding discrimination between these polymerases on model DNA templates is complicated by the fact that multiple oligomeric species are influenced by concentration and temperature. Temperature-dependent fluorescence anisotropy equilibrium binding experiments were used to separate discrete binding events for the formation of trimeric Dpo1 and dimeric Dpo4 complexes on DNA. The associated equilibria are found to be temperature-dependent, generally leading to improved binding at higher temperatures for both polymerases. At high temperatures, DNA binding of Dpo1 monomer is favored over binding of Dpo4 monomer, but binding of Dpo1 trimer is even more strongly favored over binding of Dpo4 dimer, thus providing thermodynamic selection. Greater processivities of nucleotide incorporation for trimeric Dpo1 and dimeric Dpo4 are also observed at higher temperatures, providing biochemical validation for the influence of tightly bound oligomeric polymerases. These results separate, quantify, and confirm individual and sequential processes leading to the formation of oligomeric Dpo1 and Dpo4 assemblies on DNA and provide for a concentration- and temperature-dependent discrimination of binding undamaged DNA templates at physiological temperatures.

Archaeal orc1/cdc6 proteins.

The initiation of DNA replication in most archaeal genomes is mediated by proteins related to eukaryotic Orc1 and Cdc6. Archaeal replication origins have been mapped and their interactions with Orc1/Cdc6 proteins have been characterized at the biochemical level. Structural and biophysical studies have revealed the basic rules of sequence recognition by archaeal initiators.

MCM structure and mechanics: what we have learned from archaeal MCM.

Minichromosome maintenance (MCM) complexes have been identified as the primary replicative helicases responsible for unwinding DNA for genome replication. The focus of this chapter is to discuss the current structural and functional understanding of MCMs and their role at origins of replication, which are based mostly on the studies of MCM proteins and MCM complexes from archaeal genomes.

The GINS complex: structure and function.

Eukaryotic chromosomal DNA replication is controlled by a highly ordered series of steps involving multiple proteins at replication origins. The eukaryotic GINS complex is essential for the establishment of DNA replication forks and replisome progression. GINS is one of the core components of the eukaryotic replicative helicase, the CMG (Cdc45-MCM-GINS) complex, which unwinds duplex DNA ahead of the moving replication fork. Eukaryotic GINS also links with other key proteins at the fork to maintain an active replisome progression complex. Archaeal GINS homologues play a central role in chromosome replication by associating with other replisome components. This chapter focuses on the molecular events related with DNA replication initiation, and summarizes our current understanding of the function, structure and evolution of the GINS complex in eukaryotes and archaea.

Synergic and opposing activities of thermophilic RecQ-like helicase and topoisomerase 3 proteins in Holliday junction processing and replication fork stabilization.

RecQ family helicases and topoisomerase 3 enzymes form evolutionary conserved complexes that play essential functions in DNA replication, recombination, and repair, and in vitro, show coordinate activities on model recombination and replication intermediates. Malfunctioning of these complexes in humans is associated with genomic instability and cancer-prone syndromes. Although both RecQ-like and topoisomerase 3 enzymes are present in archaea, only a few of them have been studied, and no information about their functional interaction is available. We tested the combined activities of the RecQ-like helicase, Hel112, and the topoisomerase 3, SsTop3, from the thermophilic archaeon Sulfolobus solfataricus. Hel112 showed coordinate DNA unwinding and annealing activities, a feature shared by eukaryotic RecQ homologs, which resulted in processing of synthetic Holliday junctions and stabilization of model replication forks. SsTop3 catalyzed DNA relaxation and annealing. When assayed in combination, SsTop3 inhibited the Hel112 helicase activity on Holliday junctions and stimulated formation and stabilization of such structures. In contrast, Hel112 did not affect the SsTop3 DNA relaxation activity. RecQ-topoisomerase 3 complexes show structural similarity with the thermophile-specific enzyme reverse gyrase, which catalyzes positive supercoiling of DNA and was suggested to play a role in genome stability at high temperature. Despite such similarity and the high temperature of reaction, the SsTop3-Hel112 complex does not induce positive supercoiling and is thus likely to play different roles. We propose that the interplay between Hel112 and SsTop3 might regulate the equilibrium between recombination and anti-recombination activities at replication forks.

Rapid progress of DNA replication studies in Archaea, the third domain of life.

Archaea, the third domain of life, are interesting organisms to study from the aspects of molecular and evolutionary biology. Archaeal cells have a unicellular ultrastructure without a nucleus, resembling bacterial cells, but the proteins involved in genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of Eukaryota. Therefore, archaea provide useful model systems to understand the more complex mechanisms of genetic information processing in eukaryotic cells. Moreover, the hyperthermophilic archaea provide very stable proteins, which are especially useful for the isolation of replisomal multicomplexes, to analyze their structures and functions. This review focuses on the history, current status, and future directions of archaeal DNA replication studies.

The sub-cellular localization of Sulfolobus DNA replication.

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

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.

Characterization of DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis.

In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3' → 5' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.