The active site of the rolling circle replication protein Rep75 is involved in site-specific nuclease, ligase and nucleotidyl transferase activities

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via a rolling circle mechanism. The protein Rep75, encoded by this plasmid, exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin sequence. In addition, Rep75 catalyses a site-specific nucleotidyl terminal transferase (NTT) activity, e.g. it can transfer one AMP or dAMP (from ATP or dATP) to the 3'-OH of an oligonucleotide corresponding to the left part of the nicking site. The Rep75 sequence contains a motif similar to the active-site motifs of Rep proteins from the PhiX174/pC194 superfamily. We show here that the tyrosine present in this motif is indeed essential for DNA cleavage by Rep75, but is dispensable for its NTT activity. However, a nearby arginine, which is not required for DNA cleavage, is involved in both NTT and closing, indicating that the same active site is involved in the NC and NTT activities of Rep75. For both NTT and NC, the G residue in 3' of the nicking site is essential, whereas the A residue in 5' is dispensable for NC, despite its conservation in RC plasmids of the PhiX174/pC194 superfamily. The NTT and closing activities have an optimal temperature lower than the nicking activity. These data indicate that the three reactions catalysed by Rep75 can be uncoupled, although they share part of their mechanisms. Finally, we show that NC is inhibited by ATP or dATP at concentrations that promote NTT. We propose a model in which the NTT activity of Rep75 plays a role in the regulation of pGT5 replication in vivo.     

A rolling circle replication initiator protein with a nucleotidyl-transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli , and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking-closing activity in vitro on single-stranded oligonucleotides containing the putative double-stranded replication origin sequence of pGT5. Gel shift analyses on single-stranded oligonucleotides indicate that Rep75 recognizes the single-stranded DNA region upstream of the nicking site via non-covalent interaction and remains covalently linked to the 5'-phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3'-OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.     

Control of DNA topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus

Plasmid topology varies transiently in hyperthermophilic archaea during thermal stress. As in mesophilic bacteria, DNA linking number (Lk) increases during heat shock and decreases during cold shock. Despite this correspondence, plasmid DNA topology and proteins presumably involved in DNA topological control in each case are different. Plasmid DNA in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typical of bacteria, eukaryotes and mesophilic archaea. We have analysed the regulation of DNA topological changes during thermal stress in Sulfolobus islandicus (kingdom Crenarchaeota), which harbours two plasmids, pRN1 and pRN2. In parallel with plasmid topological variations, we analysed levels of reverse gyrase, topoisomerase VI (Topo VI) and the small DNA-binding protein Sis7, as well as topoisomerase activities in crude extracts during heat shock from 80 degrees C to 85-87 degrees C, and cold shock from 80 degrees C to 65 degrees C. Quantitative changes in reverse gyrase, Topo VI and Sis7 were not significant. In support of this, inhibition of protein synthesis in S. islandicus during shocks did not alter plasmid topological dynamics, suggesting that an increase in topoisomerase levels is not needed for control of DNA topology during thermal stress. A reverse gyrase activity was detected in crude extracts, which was strongly dependent on the assay temperature. It was inhibited at 65 degrees C, but was greatly enhanced at 85 degrees C. However, the intrinsic reverse gyrase activity did not vary with heat or cold shock. These results suggest that the control of DNA topology during stress in Sulfolobus relies primarily on the physical effect of temperature on topoisomerase activities and on the geometry of DNA itself. Additionally, we have detected an enhanced thermoresistance of reverse gyrase activities in cultures subject to prolonged heat shock (but not cold shock). This acquired thermotolerance at the enzymatic level is abolished when cultures are treated with puromycin, suggesting a requirement for protein synthesis.     

Identification of a Type I Topoisomerase Activity from a Mesophilic Archaeon Methanosarcina barkeri

We have identified a type I DNA Topoisomerase from a mesophilic archaebacteria, Methanosarcina barkeri. The enzyme activity residing in a high molecular mass complex is found to be magnesium-dependent and relaxes negatively supercoiled DNA. The properties of the topoisomerase activity were detected by using the technique of transfering radioactivity from³²P labelled DNA to the protein. In presence of the enzyme, nicks are generated in the supercoiled DNA and the enzyme becomes covalently attached to the DNA. A tyrosine residue of the enzyme was found to be responsible for the covalent linkage.     

Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5′-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.     

Bacillus cereus DNA topoisomerase I and IIIalpha: purification, characterization and complementation of Escherichia coli TopoIII activity

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIα (bcTopo I, bcTopo IIIα), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg²⁺ but does not possess an efficient DNA decatenation activity. bcTopo IIIα is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg²⁺ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIα are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.     

55.2, a Phage T4 ORFan Gene,Encodes an Inhibitor of Escherichia coli Topoisomerase I and Increases Phage Fitness

Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA) gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield.     

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.     

DNA topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses

In order to address the dynamics of DNA topology in hyperthermophilic archaea, we analysed the topological state of several plasmids recently discovered in Thermococcales and Sulfolobales. All of these plasmids were from relaxed to highly positively super-coiled in vitro, i.e. they exhibited a significant linking excess compared to the negatively supercoiled plasmids from mesophilic organisms (both Archaea and Bacteria). In the two archaeai orders, plasmid linking number (Lk) decreased as growth temperature was lowered from its optimal value, i.e. positively super-coiled plasmids were relaxed whereas relaxed plasmids became negatively supercoiled. Growth temperatures above the optimum correlated with higher positive supercoiling in Sulfolobales (Lk increase) but with relaxation of positive supercoils in Thermococcus sp. GE31. The topological variation of plasmid DNA isolated from cells at different growth phases were found to be species specific in both archaeai orders. In contrast, the direction of topological variation under temperature stress was the same, i.e. a heat shock correlated with an increase in plasmid positive supercoiling, whilst a cold shock induced negative supercoiling. The kinetics of these effects were analysed in Sulfolobales. In both temperature upshift (from 80 to 85C) and downshift (from 80 to 65C), a transient sharp variation of Lk occurred first, and then DNA supercoiling progressively reached levels typical of steady-state growth at the final temperature. These results indicate that DNA topology can change with physiological states and environmental modifications in hyperthermophilic archaea.     

Pea chloroplast topoisomerase I: purification,characterization, and role in replication

A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl₂. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

Comparison of plasmid DNA topology among mesophilic and thermophilic eubacteria and archaebacteria.

Several plasmid DNAs have been isolated from mesophilic and thermophilic archaebacteria. Their superhelical densities were estimated at their host strain's optimal growth temperature, and in some representative strains, the presence of reverse gyrase activity (positive DNA supercoiling) was investigated. We show here that these plasmids can be grouped in two clusters with respect to their topological state. The group I plasmids have a highly negatively supercoiled DNA and belong to the mesophilic archaebacteria and all types of eubacteria. The group II plasmids have DNA which is close to the relaxed state and belong exclusively to the thermophilic archaebacteria. All archaebacteria containing a relaxed plasmid, with the exception of the moderately thermophilic methanogen Methanobacterium thermoautotrophicum Marburg, also exhibit reverse gyrase activity. These findings show that extrachromosomal DNAs with very different topological states coexist in the archaebacterial domain.     

The Bloom's syndrome helicase stimulates the activity of human topoisomerase IIIalpha

Bloom’s syndrome (BS) is a disorder associated with chromosomal instability and a predisposition to the development of cancer. The BS gene product, BLM, is a DNA helicase of the RecQ family that forms a complex in vitro and in vivo with topoisomerase IIIα. Here, we show that BLM stimulates the ability of topoisomerase IIIα to relax negatively supercoiled DNA. Moreover, DNA binding analyses indicate that BLM recruits topoisomerase IIIα to its DNA substrate. Consistent with this, a mutant form of BLM that retains helicase activity, but is unable to bind topoisomerase IIIα, fails to stimulate topoisomerase activity. These results indicate that a physical association between BLM and topoisomerase IIIα is a prerequisite for their functional biochemical interaction.     

Characterization of the Nucleoid-associated Protein YejK

Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait. The purified protein likely exists in a monomer-dimer equilibrium in solution and can form tetramers. Cross-linking of the protein bound to DNA suggests that the active form could be either a dimer or tetramer. YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12–14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds. YejK binds RNA, but not single-stranded DNA, with 65% of the avidity with which it binds DNA. However, cells deleted for yejK do not show defects in either RNA or protein synthesis. YejK interacts with all the subunits of both DNA gyrase and topoisomerase IV and has measurable effects on their activities. In the presence of YejK, relaxation of negatively supercoiled DNA by topoisomerase IV becomes distributive, whereas relaxation of positively supercoiled DNA is stimulated. Relaxation of negatively supercoiled DNA by DNA gyrase is inhibited, whereas the extent of supercoiling of relaxed DNA is limited. A YejK-GFP chimera is an effective marker for the nucleoid in live cell imaging.     

High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=     

Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA

Genetic evidence suggests that the Bacillus subtilis lrpC gene product participates in cell growth and sporulation. The purified LrpC protein, which has a predicted molecular mass of 16.4 kDa, is a tetramer in solution. LrpC binds with higher affinity (K_app ~ 80 nM) to intrinsically curved DNA than to non-curved DNA (K_app ~ 700 nM). DNase I footprinting and the supercoiling of relaxed circular plasmid DNA in the presence of topoisomerase I revealed that LrpC induces DNA bending and constrains DNA supercoils in vitro. The LrpC protein cooperatively increases DNA binding of the bona fide DNA-binding and DNA-bending protein Hbsu. LrpC forms inter- and intramolecular bridges on linear and supercoiled DNA molecules, resulting in a large network and DNA compactation. Collectively, these findings suggest that LrpC is an architectural protein and that its activities could provide a means to modulate DNA transactions.     

High-throughput assays for DNA gyrase and other topoisomerases

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.     

DNA bending,compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus

Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea Sulfolobus. Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments.