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J N Reeve

Publications and source records attributed to J N Reeve.

At least 19 recordsLinked to original sources
PubMed1999

Archaeal nucleosome positioning sequence from Methanothermus fervidus.

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.

Archaea↗
PubMed1999

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.

DNA, Archaeal↗
PubMed1999

Methanobacterium thermoautotrophicum RNA polymerase and transcription in vitro.

RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum DeltaH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the M. thermoautotrophicum RNAP and TFB has been demonstrated.

Amino Acid Sequence↗
PubMed1999

Archaeal nucleosome positioning by CTG repeats.

DNA shape recognition determines the preferred binding sites for sequence-independent DNA binding proteins, and here we document that archaeal histones assemble archaeal nucleosomes in vitro centered preferentially within (CTG)6 and (CTG)8 repeats, close to junctions with flanking mixed-sequence DNA. Archaeal nucleosomes were not positioned by (CTG)4-, (CTG)5-, or (CTG)3AA(CTG)3-containing DNA sequences. The features of CTG repeat-containing sequences that direct eucaryal nucleosome positioning may also be similarly recognized by archaeal histones.

Archaea↗
PubMed1998

Thermodynamic stability of archaeal histones.

The temperature, salt, and pH dependencies of unfolding of four recombinant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyA1 from the hyperthermophiles Methanothermus fervidus and Pyrococcus strain GB-3a) have been determined by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). The thermal unfolding of these proteins is > 90% reversible, with concentration-dependent apparent Tm values and asymmetric unfolding transitions that are fit well by a two-state unfolding model in which a histone dimer unfolds to two random coil monomers. rHPyA1 dimers are stable in the absence of salt, whereas rHMfA, rHMfB, and rHFoB dimers unfold at 20 degrees C and pH 2 in solutions containing < 200 mM, < 400 mM, and < 1.5 M KCl, respectively. rHMfA, rHMfB, and rHFoB also experience significant cold denaturation in low salt concentrations and at low pH. The midpoint of thermal unfolding of a 1 M protein solution (T degree value) and the temperature dependency of the free energy of unfolding have been established for each histone, and both parameters correlate with the growth temperature of the originating archaeon. The changes in heat capacity upon unfolding are similar for the four histones, indicating that enhanced thermostability is not obtained by altering the curvature of the stability curve. Rather, the stability curves for the histones from the hyperthermophiles are displaced vertically to higher energies and laterally to higher Tmax values relative to the stability curve for rHFoB. The maximal free energies of unfolding for rHFoB, rHMfA, rHMfB, and rHPyA1 are 7.2, 15.5, 14.6, and 17.2 kcal/mol at 32, 35, 40, and 44 degrees C, respectively. T degree values for rHFoB, rHMfA, rHMfB, and rHPyA1 are 75, 104, 113, and 114 degrees C, respectively, at pH 5 in 0.2 M KCl. Structural features within the conserved histone fold that might confer these stability differences are discussed.

Amino Acid Sequence↗
PubMed1998

NMR structure and comparison of the archaeal histone HFoB from the mesophile Methanobacterium formicicum with HMfB from the hyperthermophile Methanothermus fervidus.

The solution-state structure of the recombinant archaeal histone rHFoB, from the mesophile Methanobacterium formicicum, has been determined by two- and three-dimensional (3D) proton homonuclear correlated nuclear magnetic resonance (NMR) methods. On the basis of 951 nuclear Overhauser effect (NOE)-derived distance restraints, rHFoB monomers form the histone fold and assemble into symmetric (rHFoB)2 dimers that have a structure consistent with assembly into archaeal nucleosomes. rHFoB exhibits approximately 78% sequence homology with rHMfB from the hyperthermophile Methanothermus fervidus, and the results obtained demonstrate that these two proteins have very similar 3D structures, with a root-mean-square deviation for backbone atoms of 0.65 +/- 0.13 A2. (rHFoB)2 dimers however unfold at lower temperatures and require a higher salt environment for stability than (rHMfB)2 dimers, and comparing the structures, we predict that these differences result from unfavorable surface-located ionic interactions and a larger, more solvent-accessible cavity adjacent to residue G36 in the hydrophobic core of (rHFoB)2.

Amino Acid Sequence↗
PubMed1998

Diversity of prokaryotic chromosomal proteins and the origin of the nucleosome.

All cells employ architectural proteins to confine and organize their chromosomes, and to prevent the otherwise thermodynamically favored collapse of concentrated DNA into compact structures. To accomplish this, prokaryotes have evolved a variety of phylogenetically unrelated, small, basic, sequence-independent DNA-binding proteins that include histones in Euryarchaeota, and members of the HU family in many Bacteria. In contrast, virtually, all Eukarya employ histones, and recently a metabolism-based hypothesis proposed that the eukaryal nucleus originated from a hydrogen-consuming, histone-containing Archaeon. Histones may have prevailed during the evolution of the Eukarya because of their extended interactions with DNA and, as noted, the histone fold now exists not only in histones but also as a structural motif in eukaryal transcription factors.

Amino Acid Sequence↗
PubMed1998

Archaeal histone stability, DNA binding, and transcription inhibition above 90 degrees C.

The DNA binding and compacting activities of the recombinant (r) archaeal histones rHMfA and rHMfB from Methanothermus fervidus, and rHPyA1 from Pyrococcus species GB-3a, synthesized in Escherichia coli, have been shown to be completely resistant to incubation for 4h at 95 degrees C in the presence of 1M KCl. Continued incubation of rHMfA and rHMfB at 95 degrees C resulted in a gradual loss of these activities, and rHMfA and rHMfB lost activity more rapidly at 95 degrees C when the salt environment was reduced to 200 mM K Cl. rHPyA1, in contrast, retained full activity even after a 60-h incubation at 95 degrees C in 1 M KCl, and reducing the salt concentration did not affect the heat resistance of rHPyA1. rHPya1-DNA complexes remained intact at 100 degrees C, and rHPyA1 bound to the template DNA in in vitro transcription reaction mixtures assembled using Pyrococcus furiosus components at 90 degrees C. Transcription in vitro from the P. furiosus gdh promoter was reduced by rHPyA1 binding, in a manner that was dependent on the histone-to-DNA ratio and on the topology of the DNA template. Transcription from circular templates was more sensitive to rHPyA1 binding than transcription from a linear template, consistent with rHPyA1 binding introducing physical barriers to transcription and causing changes in the topology of circular templates that also reduced transcription.

Amino Acid Sequence↗
PubMed1998

Histones and nucleosomes in Archaea and Eukarya: a comparative analysis.

Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion. Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3 + H4)2 tetramer.

Amino Acid Sequence↗
PubMed1997

Archaeal nucleosomes.

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect approximately 60 bp of DNA and multiples of approximately 60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.

Archaea↗
PubMed1997

Large scale preparation of positively supercoiled DNA using the archaeal histone HMf.

A technique to prepare relatively large quantities (>/=100 microg) of highly positively supercoiled DNA is reported. This uses a recombinant archaeal histone (rHMfB) to introduce toroidal supercoils, and an inexpensive chicken blood extract to relax unrestrained superhelical tension. Preparation of positively supercoiled pUC19 DNA molecules, >50% of which have linking number changes ranging from+8 to+17, is demonstrated. Advantages include the high degree of positive supercoiling that can be achieved, control over the extent of supercoiling, easy production of relatively large quantities of supercoiled DNA, and low cost.

Animals↗
PubMed1997

DNA binding and nuclease protection by the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus.

The DNA-binding and nuclease-protection properties of the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus have been shown to be consistent with the formation of nucleosome-like structures (NLS). These proteins bind to DNA molecules as short as 20 bp and form complexes that protect DNA fragments from micrococcal nuclease (MNase) digestion that are 30 bp, approximately 60 bp and multiples of approximately 60 bp in length. The sequences of 49 of the approximately 60-bp DNA fragments protected from MNase digestion by HMfA have been determined and their intrinsic curvatures calculated. A circular permutation gel mobility-shift assay was used to determine directly the curvatures for five of these sequences. HMfA bound to intrinsically curved and noncurved DNAs, but exhibited a slight preference for the model curved DNA in binding competitions with a model noncurved DNA. The results obtained are consistent with the concept that the archaeal NLS is analogous, and possibly homologous, to the central core of the eukaryal nucleosome formed by a histone (H3 + H4)2 tetramer.

Base Sequence↗
PubMed1997

Differential effects of DNA supercoiling on radical-mediated DNA strand breaks.

Supercoiling is an important feature of DNA physiology in vivo. Given the possibility that the reaction of genotoxic molecules with DNA is affected by the alterations in DNA structure and dynamics that accompany superhelical tension, we have investigated the effect of torsional tension on DNA damage produced by five oxidizing agents: gamma-radiation, peroxynitrite, Fe2+/ EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. With positively supercoiled plasmid DNA prepared by a recently developed technique, we compared the quantity of strand breaks produced by the five agents in negatively and positively supercoiled pUC19. It was observed that strand breaks produced by gamma-radiation, peroxynitrite, and Fe2+/EDTA/H2O2 were insensitive to DNA superhelical tension. These results are consistent with a model in which chemicals that generate highly reactive intermediates (e.g., hydroxyl radical), but do not interact directly with DNA, will be relatively insensitive to the changes in DNA structure and dynamics caused by superhelical tension. In the case of Fe2+ and Cu2+, metals that bind to DNA, only Cu2+/H2O2 proved to be sensitive to DNA superhelical tension. Strand breaks produced by Cu2+/H2O2 in the positively supercoiled substrate occurred at lower Cu concentrations than in negatively supercoiled DNA. Furthermore, a sigmoidal Cu2+/H2O2 damage response was observed in the negatively supercoiled substrate but not in positively supercoiled DNA. The results with Cu2+ suggest that the redox activity, DNA binding orientation, or DNA binding affinity of Cu1+ or Cu2+ is sensitive to superhelical tension, while the results with the other oxidizing agents warrant further investigation into the role of supercoiling in base damage.

DNA↗
PubMed1997

Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics.

The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.

Anaerobiosis↗