Oligoribonucleotide circularization by 'template-mediated' ligation with T4 RNA ligase: synthesis of circular hammerhead ribozymes

Circular hammerhead ribozymes were synthesized from linear oligoribonucleotides using T4 RNA ligase. Some of the precursors could not be efficiently circularized under standard conditions. For these molecules, the use of a DNA template allowed their efficient circularization. The template was designed to prevent the precursor from folding into an unsuitable structure. The template allowed circular ribozymes as small as 15 nucleotides in length to be efficiently synthesized at concentrations as high as 50 microM in the ligation reaction. The circular products retained their biological activity.     

DNA binding properties of a chemically synthesized DNA binding domain of hRFX1

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (approximately 90 mg) of pure compound. Preliminary two-dimensional1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (K a = 4 x 10(5)/M/s), a dissociation constant (K d = 6 x 10(-2)/s) and an equilibrium dissociation constant (K D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degreesC, illustrating stabilization of the double-stranded conformation induced by the peptide.     

Site-specific endonuclease from thermophilic Bacillus species MK strain is isoschizomer of SalI

Screening of thermophilic bacterial strains revealed a strain containing site-specific endonuclease BspMKI. This endonuclease was purified to functional homogeneity during sequential chromatographic steps. The enzyme recognizes sequence 5'-G decreases TCGAC-3' on DNA molecule and is isoschizomer of endonuclease SalI. The molecular mass of BspMKI is about 45 kD. The enzyme is maximally active at 55 degrees C and MRB (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol) is the optimal buffer. The enzyme is highly stable and retains its activity during two weeks at room temperature.     

Telomerase protein rather than its RNA is the target of phosphorothioate-modified oligonucleotides

Human telomerase is a ribonucleoprotein which uses its internal RNA moiety as a template for telomeric DNA synthesis. This enzyme is up-regulated in most malignant tumors and is therefore considered as a possible cancer target. Here we examined the effects of differently modified oligomers on telomeraseactivity from HL-60 cell extracts (TRAP-ezetrade mark assay). Phosphorothioate-modified oligonucleotides (PS-ODNs) inhibited telomerase activity at subnanomolar concen-trations and proved to be more efficient than peptide nucleic acids. In contrast to all the investigated oligomers, PS-ODNs were found to bind to the protein motif of telomerase called the primer binding site but poorly to its RNA. This is suggested by kinetic investigations demonstrating a competitive interaction of PS-ODNs and TS primer at the primer binding site. The K m value of the TS primer was 10.8 nM, the K i value of a 20mer PS-ODN was 1.6 nM. When the TS primer was PS-modified a striking increase in the telomerase activity was found which correlates with the number of phosphodiesters replaced. The K m value of a completely PS-modified TS primer was 0.56 nM. Based on these results the design of chimeric ODNs is proposed consisting of a 5'-PS-modified part targeting the primer binding site and a 3'-terminus part targeting the telomerase RNA.     

Kinetics of the light-induced proton translocation associated with the pH-dependent formation of the metarhodopsin I/II equilibrium of bovine rhodopsin

The kinetics of the formation of the metaII (MII) state of bovine rhodopsin was investigated by time-resolved electrical and absorption measurements with rod outer segment (ROS) fragments. Photoexcitation leads to proton transfer in the direction from the cytosolic to the intradiscal side of the membrane, probably from the Schiff base to the acceptor glutamate 113. Two components of comparable amplitude are required to describe the charge movement with exponential times of 1.1 (45%) and 3.0 ms (55%) (pH 7.8, 22 degreesC, 150 mM KCl). The corresponding activation energies are 86 and 123 kJ/mol, respectively (150 mM KCl). The time constants and amplitudes depend strongly on pH. Between pH 7.1 and 3.8 the kinetics becomes much faster, with the faster and slower components accelerating by factors of about 8 and 2, respectively. Complementary single-flash absorption experiments at 380 nm and 10 degreesC show that the formation of MII also occurs with two components with similar time constants and pH dependence. This suggests that both signals monitor the same molecular events. The pH dependence of the two apparent time constants and amplitudes of the optical data can be described well over the pH range 4-7.5 by two coupled equilibria between MI and two isochromic MII species MIIa and MIIb: MI MIIa(380) MIIb(380), with k0 proportional to the proton concentration. This model implies that deprotonation of the Schiff base and proton uptake are tightly coupled in ROS membranes. Models with k2 proportional to the proton concentration cannot describe the data. Photoreversal of MII by blue flashes (420 nm) leads to proton transfer in a direction opposite to that of the signal associated with MII formation. In this transition the Schiff base is reprotonated, most likely from glutamate 113. At pH 7.3, 150 mM KCl, 22 degreesC, this electrical charge reversal has an exponential time constant of about 30 ms and is about 10 times slower than the forward charge motion.     

Template-directed photoligation of oligodeoxyribonucleotides via 4-thiothymidine

Non-enzymatic, template-directed ligation of oligonucleotides in aqueous solution has been of great interest because of its potential synthetic and biomedical utility and implications for the origin of life. Though there are many methods for template-directed chemical ligation of oligonucleotides, there are only three reported photochemical methods. In the first report, template-directed photoligation was effected by cyclobutane dimer formation between the 5'- and 3'-terminal thymidines of two oligonucleotides with >290 nm light, which also damages DNA itself. To make the photochemistry of native DNA more selective, we have replaced the thymidine at the 5'-end of one oligonucleotide with 4-thiothymidine (s4T) and show that it photoreacts at 366 nm with a T at the 3'-endof another oligonucleotide in the presence of a complementary template. When a single mismatch is introduced opposite either the s4T or its adjoining T, the ligation efficiency drops by a factor of five or more. We also show that by linking the two ends of the oligonucleotides together, photoligation can be used to form circular DNA molecules and to 'photopadlock' circular DNA templates. Thus, s4T-mediated photo-ligation may have applications to phototriggered antisense-based or antigene-based genetic tools, diagnostic agents and drugs, especially for those situations in which chemical or enzyme-mediated ligation isundesirable or impossible, for example inside a cell.     

Kinetic mechanism for the sequential binding of two single-stranded oligodeoxynucleotides to the Escherichia coli Rep helicase dimer

Escherichia coli Rep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss-oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P2S dimer). We show that ss-DNA binding to the P2S Rep dimer to form the doubly ligated P2S2 dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k1 = approximately 2 x 10(6) M-1 s-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4 degrees C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P2S2 Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.     

DNA-Protein crosslinks induced by nickel compounds in isolated rat renal cortical cells and its antagonism by specific amino acids and magnesium ion

Suspensions of isolated renal cortical cells in modified Krebs-Henseleit buffer (pH 7.4) were incubated with nickel chloride, nickel acetate, nickel sulfate, and nickel subsulfide (0-2 mM) at 37 degreesC for 2 h. A significant increase (63%) in DNA-protein crosslinks was observed at 2 mM nickel sulfate, whereas nickel subsulfide induced a significant increase in such crosslinks beginning at 0.5 mM concentration and a maximum increase of 200% of the control value reached at 2 mM concentration. No significant reduction in viability of renal cortical cells (as measured by trypan blue exclusion) was observed due to these nickel compounds at any concentration used. In the second series of experiments, coincubation of nickel subsulfide (2 mM) with l-histidine (8 or 16 mM), l-cysteine (4 or 8 mM), or l-aspartic acid (8 or 24 mM) significantly reduced the DNA-protein crosslinks induced by 2 mM nickel subsulfide. Similarly Mg2+ (24 mM), but not Ca2+ (24 mM), was able to antagonize nickel subsulfide-induced increase in DNA-protein crosslinks. High extracellular levels of Mg2+ and these amino acids significantly decreased the accumulation of Ni2+ from nickel subsulfide in renal cortical cells. Furthermore, these amino acids at high concentrations significantly inhibited the binding of Ni2+ from nickel subsulfide to deproteinized DNA from renal cortical cells, whereas such inhibition due to Mg2+ was close to significant (0.1 > p > 0.05). In vitro exposures of renal cortical cells to nickel subsulfide (0-2 mM) increased the formation of reactive oxygen species in concentration-dependent manner. Furthermore, coincubation of 2 mM nickel subsulfide with either catalase, dimethylthiourea, mannitol, or vitamin C at 37 degreesC for 2 h resulted in a significant decrease of nickel subsulfide-induced formation of DNA-protein crosslinks, suggesting that nickel subsulfide-induced DNA-protein crosslink formation in isolated rat renal cortical cells is caused by the formation of reactive oxygen species. The potent protective effects of these specific amino acids and Mg2+ against nickel subsulfide-induced DNA-protein crosslink formation in isolated renal cortical cells are due to reduction of cellular uptake of Ni2+ and inhibition of the binding of Ni2+ to deproteinized DNA.     

Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.     

Capillary gel electrophoresis as a method to determine ligation efficiency

A capillary gel electrophoresis (CGE) method is described for detection of the formation of circular DNA ligation products as an aid in the prediction of ligated DNA competent cell transformation efficiency. The separation is based upon the differences in the relative migrations of linear and circular DNA molecules of the same size. In CGE, circular ligation products are shifted significantly from linear DNA fragments of comparable size (to 40-42 min from 32-33 min migration time) in the presence of an intercalating dye. CGE separation and detection of circularized DNA can be correlated with transformation efficiencies of > 10(6) colony-forming units (CFU, colonies/micrograms/ml) or the high efficiency desired for phagemid display and cell expression libraries. CGE has several advantages over slab gel electrophoresis: (i) only a minute quantity (approximately 250 CFU or 0.02%) of the total library is sacrificed for analysis, (ii) verification of the circularized ligation products is easier by CGE, and (iii) CGE analysis of ligation success can be accomplished in less than 2 h, prior to transforming competent cells.     

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.     

Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana

Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95 degreesC, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106 degreesC. Both enzymes are characterized by a high level of activity (high Vmax value and low apparent Km value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the Vmax values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106 degreesC and 26 min at 110 degreesC. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.     

Physico-chemical and antigenic properties of tetanus and diphtheria toxoids and steps towards improved stability

Physico-chemical, antigenic and immunogenic properties may be altered during microencapsulation of antigens and their release from poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres. Here, the physico-chemical, conformational and antigenic stability of tetanus and diphtheria toxoids was studied in aqueous solutions stressed by elevated temperature and the presence of lactic and glycolic acids. Further, the stabilising effect of albumin was investigated. The analytical tools used were fluorimetry, circular dichroism spectroscopy, turbidimetry, electrophoresis and ELISA. Elevated temperatures altered the physico-chemical and antigenic properties of the toxoids to a greater extent than the acids (50 mM) did. Substantial unfolding and chemical changes of tryptophan were observed upon 1-4 weeks of incubation at 60 degreesC. At 4 degreesC, only minor conformational changes were observed, even in the presence of the acids. Furthermore, 40% of the tetanus toxoid antigenicity was lost after 7 days at 37 degreesC. This loss increased in the presence of the acids. At 60 degreesC, the antigenicity had completely vanished. Very importantly, 0.5% albumin preserved the tetanus antigenicity over 6 weeks' incubation at 37 degreesC, regardless of the presence of glycolic acid. This qualifies albumin as potential stabilising additive for toxoid loaded poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres.     

Targeting of nucleic acid junctions: addressing to a branch point an oligodeoxynucleotide conjugated with an intercalator

It is possible to enhance targeting of a DNA stem flank domain with a complementary DNA when it is conjugated with diphenyl ether at the branch point. The nucleoside 2'-deoxy-5-methyl- N 4-(4-phenoxyphenyl)cytidine (5) was synthesized from thymidineby tritylation, acetylation, amination via 2,4, 6-trimethylbenzenesulfonyl activation and subsequent de-protection. When a three-way junction is formed with a bulged nucleoside 5 at the branch point, the thermal melting temperature was increased by 9 degreesC when compared with wild-type DNA. When hybridizing to one of the flanks at a stem allowing coaxial stacking to the stem, modification at the branch point resulted in DeltaTm= 5.8 degreesC. For targeting to RNA the results were more ambiguous. RNase H activity was observed in some cases when an intercalating aromatic ring was addressed at the branch point. RNase H activity was observed even for a short 7mer ODN.     

X-ray crystal structures of half the human papilloma virus E2 binding site: d(GACCGCGGTC)

The X-ray crystal structure of the DNA decamer d(GACCGCGGTC), containing half the human papilloma virus E2 binding site, has been solved from two crystals grown at different ionic conditions (50 mM MgCl2and 50 mM spermine or 1.56 mM MgCl2and 1.56 mM spermine). Despite the variation in salt concentration, the two DNA structures are in a very similar, A-type DNA conformation, with helical axes curving towards the major groove. Although the salt concentrations do not effect the helical parameters or hydration to a large degree, there is a change in the overall helical curvature; 18 degrees and 31 degrees for the low and high salt structures, respectively. This curvature appears to be sequence specific and biologically relevant when compared with similar DNA structures, including the E2 binding site of a protein-DNA complex.     

Inhibition of anchorage-independent growth of ras-transformed cells on polyHEMA surface by antisense oligodeoxynucleotides directed against K-ras

We examined the effect of antisense oligodeoxynucleotides (AS ODNs) directed against v-Ki-ras on the anchorage-independent growth of v-Ki-ras-transformed rat fibroblasts using plates coated with an antiadhesive polymer, poly(2-hydroxyethyl methacrylate) (polyHEMA). Among AS ODNs tested, 17mer AS ODN centered around codon 12 of v-Ki-ras inhibited the v-Ki-Ras expression and v-Ki-Ras-induced anchorage-independent growth, while its sense sequence did not affect it. Using polyHEMA plates, the direct addition of AS ODNs to the cells growing anchorage-independently and various treatment schedules of AS ODNs are now available to identify the most desirable conditions for AS ODNs treatment.     

Synthesis and properties of covalently-closed DNA duplexes containing pyrophosphate and and substituted pyrophosphate internucleotide groups

A new method for the efficient synthesis of covalently closed DNA duplexes (DNA dumbbells) and the introduction of pyrophosphate and substituted pyrophosphate internucleotide groups into their structure is proposed. The method is based on chemical ligation in DNA duplexes that are formed by a polynucleotide the ends of which are brought together due to the introduction of the minihairpin structure [sequence: see text]. DNA dumbbells containing a pyrophosphate (substituted pyrophosphate) group result from the interaction as being between the 3'-terminal phosphate (methylphosphate) group of the polynucleotide and the 5'-terminal phosphate group of deoxyguanosine of the minihairpin sequence, which flanks the polynucleotide from the 5' end. 1-Ethyl-3-(3'-dimethylaminopropyl) carbodiimide was used as a condensing agent. The yield of covalently closed 42-mer DNA duplexes containing a pyrophosphate group was 98%, that of duplexes with a substituted pyrophosphate group was 25%. The reactivity of the substituted pyrophosphate group incorporated into DNA dumbbells was studied. It is shown that the group efficiently interacts with nucleophiles in an aqueous medium at pH 8.0.     

Structural and biological stability of the human interleukin 10 homodimer

Human interleukin 10 (huIL-10) is a cytokine that regulates the synthesis of type 1 helper T cell derived cytokines such as gamma-interferon, interleukin 2, and tumor necrosis factor alpha. The potential immunosuppressive activities of huIL-10 suggest that this protein may be clinically useful for treating autoimmune diseases. Due to the potential clinical value of this cytokine, physicochemical studies have been performed regarding its association state and biological/structural stability. These studies include performing size-exclusion chromatography, chemical cross-linking, equilibrium ultracentrifugation, and circular dichroism spectroscopy. The results indicate huIL-10 is predominantly a noncovalent homodimer at neutral pH and 4 degreesC for concentrations greater than 0.003 mg/mL (0.08 microM dimer). An apparent pKa value of approximately 4.8 was calculated for both the pH-dependent subunit dissociation and pH-induced loss in MC/9 biological activity. A temperature analysis revealed a linear relationship between the percent dimer and relative MC/9 activity, thus, these results and the pH-dependent activity results suggest that the huIL-10 dimer is the active species. The GndHCl-induced unfolding of rhuIL-10, monitored by far-UV circular dichroism, revealed a unique biphasic unfolding process which contained both a subunit dissociation process (<1.6 M GndHCl) as well as the unfolding of a highly alpha-helical monomer intermediate ([GndHCl]1/2 = 3.5 M). The monomer intermediates generated with 1.6 M GndHCl or pH 2.5 retained approximately 80% and 89% of the alpha-helical content of the native protein, respectively. Although a soluble and highly helical monomer state can be generated, the observed correlation between unfolding studies and biological activity suggests the dimer is the active species. These results are consistent with both the recent observation that the three-dimensional structure of rhuIL-10 is a 2-fold symmetric homodimer and that a complex between the extracellular domain of the recombinant human IL-10 receptor and IL-10 is consistent with two IL-10 homodimers and four receptors.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.