Nucleosome-like Complex of the Histone from the Hyperthermophile Methanopyrus Kandleri (MkaH) with Linear DNA

Abstract

The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (Rw) of ≈3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw ≤ 3 revealed formation of isometric loops encompassing 71 +/- 7 bp of DNA duplex. At high values of Rw (≥9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)₂ tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.     

Negative constrained DNA supercoiling in archaeal nucleosomes

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.     

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. Received: January 22, 1998 / Accepted: February 16, 1998     

A comparison of the DNA binding properties of histone-like proteins derived from representatives of the two kingdoms of the Archaea

Abstract The nucleoid protein composition, the enhancement of DNA electrophoretic mobility, the toroidal wrapping and the helical period of DNA complexed with nucleoid proteins from species within the archaeal kingdom Euryarchaeota was shown to contrast with the composition and properties of nucleoid proteins from Sulfolobus solfataricus , a member of the archaeal kingdom Crenarchaeota. This result was seen to support the hypothesis that archaeal histones with homology to the eukaryal hi stone consensus are a diagnostic feature of the Euryarchaeota.     

The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers.

BACKGROUND: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered. RESULTS: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3/H4 tetramers, and the histone-fold archaeal protein HMf. The data unequivocally show that HMf reconstitutes are indeed organized as chromatin fibers, morphologically indistinguishable from their eukaryal counterparts. The nuclease digestion patterns revealed a clear pattern of protection at regular intervals, again similar to the patterns observed with eukaryal chromatin fibers. In addition, we studied HMf reconstitutes on mononucleosome-sized DNA fragments and observed a great degree of similarity in the internal organization of these particles and those organized by H3/H4 tetramers. A difference in stability was observed at the level of mono-, di-, and triparticles between the HMf particles and canonical octamer-containing nucleosomes. CONCLUSIONS: The in vitro reconstituted HMf-nucleoprotein complexes can be considered as bona fide chromatin structures. The differences in stability at the monoparticle level should be due to structural differences between HMf and core histone H3/H4 tetramers, i.e., to the complete absence in HMf of histone tails beyond the histone fold. We speculate that the existence of core histone tails in eukaryotes may provide a greater stability to nucleosomal particles and also provide the additional ability of chromatin structure to regulate DNA function in eukaryotic cells by posttranslational histone tail modifications.     

The V(D)J recombination signal sequence and kappa B binding protein Rc binds DNA as dimers and forms multimeric structures with its DNA ligands.

The murine DNA binding protein Rc binds to the heptamer motif of the V(D)J recombination signal sequences and to the kappa B motif of the immunoglobulin enhancer. Bacterial fusion proteins for Rc and DNA ligands of Rc form multiple protein-DNA complexes in electrophoretic mobility shift assays (EMSA). Large complexes formation is favored by an increased Rc concentration. In order to determine the architecture of these complexes, the apparent molecular weights of the protein-DNA complexes were first determined by their gel mobilities. The data suggest that Rc binds to its DNA ligands as dimers, tetramers, and multiples of tetramers. The inference that Rc binds DNA as dimers was substantiated by the formation of chimeric complexes when two electrophoretically distinguishable Rc proteins were employed in EMSA. Methylation interference experiments show that there are no contiguous protein binding sites evident in the DNA of the larger complexes. Apparently, multimerization occurs via protein-protein interactions. Such interaction was demonstrated by the formation of Rc dimers and tetramers in a chemical crosslinking experiment. Significantly, the multimerization of DNA-bound Rc could be involved in bringing the variable region gene segments together for the somatic V(D)J recombination.     

Surface histidine residue of archaeal histone affects DNA compaction and thermostability

Archaeal histone, which possesses only the core domain part of eukaryal histone, induced DNA compaction by binding to DNA. Based on structural modeling, tetramer formation by dimer-dimer interaction is considered to require two intermolecular ion pairs formed between histidine and aspartate. To examine the role of the ion pairs on DNA compaction, mutant histones were constructed and analyzed using HpkB from Thermococcus kodakaraensis KOD1 as a model protein. The mutant histones, HpkB-H50A, HpkB-H50V, and HpkB-H50G were constructed by replacing conserved surface His50 with Ala, Val, and Gly, respectively. Circular dichroism analysis indicated no significant difference between wild-type and mutants in their structures. Gel mobility shift assays showed that all mutants possessed DNA binding ability, like wild-type HpkB, however all mutants compacted DNA less efficiently than the wild-type. Moreover, all mutants could not maintain the nucleosome-like structure (compacted form of DNA) above 80 degrees C. These results suggest that surface ion pairs between His and Asp play an important role in maintenance of nucleosome structure and DNA stabilization at high temperature.     

Crystal structures of recombinant histones HMfA and HMfB from the hyperthermophilic archaeon Methanothermus fervidus

The hyperthermophilic archaeon Methanothermus fervidus contains two small basic proteins, HMfA (68 amino acid residues) and HMfB (69 residues) that share a common ancestry with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. HMfA and HMfB have sequences that differ at 11 locations, they have different structural stabilities, and the complexes that they form with DNA have different electrophoretic mobilities. Here, crystal structures are documented for recombinant (r) HMfA at a resolution of 1.55 A refined to a crystallographic R-value of 19.8 % (tetragonal form) and at 1.48 A refined to a R-value of 18.8 % (orthorhombic form), and for rHMfB at 1.9 A refined to a R-value of 18.0 %. The rHMfA and rHMfB monomers have structures that are just histone folds in which a long central alpha-helix (alpha2; 29 residues) is separated from shorter N-terminal (alpha1; 11 residues) and C-terminal (alpha3; 10 residues) alpha-helices by two loops (L1 and L2; both 6 residues). Within L1 and L2, three adjacent residues are in extended (beta) conformation. rHMfA and rHMfB assemble into homodimers, with the alpha2 helices anti-parallel aligned and crossing at an angle of close to 35 degrees, and with hydrogen bonds formed between the extended, parallel regions of L1 and L2 resulting in short beta-ladders. Dimerization creates a novel N-terminal structure that contains four proline residues, two from each monomer. As prolines are present at these positions in all archaeal histone sequences, this proline-tetrad structure is likely to be a common feature of all archaeal histone dimers. Almost all residues that participate in monomer-monomer interactions are conserved in HMfA and HMfB, consistent with the ability of these monomers to form both homodimers and (HMfA+HMfB) heterodimers. Differences in side-chain interactions that result from non-conservative residue differences in HMfA and HMfB are identified, and the structure of a (rHMfA)(2)-DNA complex is presented based on the structures documented here and modeled by homology to histone-DNA interactions in the eukaryal nucleosome.     

Soluble acidic complexes containing histones H3 and H4 in nuclei of Xenopus laevis oocytes

Oocyte nuclei of Xenopus laevis contain nucleosomal-core histones in large amounts and in a soluble, non-chromatin-bound form. Supernatant fractions (100,000 X g) from isolated nuclei are enriched in complexes containing histones H3 and H4, which are of distinct size (5.6S by sucrose gradient centrifugation, approximate molecular weight of 270,000 by gel filtration) and negatively charged (isoelectric at pH 4.4). These complexes bind to DEAE-Sephacel and can be separated from nucleoplasmin. In diverse fractionation experiments, histones H3 and H4 have been found to comigrate with a pair of polypeptides of molecular weight 110,000 that represent the most acidic major protein present in these nuclei. After enrichment by gel filtration, ion exchange chromatography and electrophoresis, this pair of acidic polypeptides has been the only nonhistone protein detected in the histone-complex fraction. We suggest that in the oocyte nucleus, large proportions of the soluble histones H3 and H4 are not contained in complexes of all four nucleosomal-core histones but are differentially associated with specific, very acidic proteins into distinct 5.6S complexes.     

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, ∼ 60 bp and multiples of ∼ 60 bp in length. The sequences of 49 of the ∼ 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. Received: August 11, 1996 / Accepted: November 12, 1996     

Recombinant expression and characterization of an extremely hyperthermophilic archaeal histone from Pyrococcus horikoshii OT3

A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65 degrees C. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90 degrees C. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.     

Co-existence of two different types of soluble histone complexes in nuclei of Xenopus laevis oocytes

The nuclear pool of soluble histones in Xenopus laevis oocytes is organized into two major types of acidic histone complexes separable by sucrose density gradient centrifugation. One type of complex sediments at 5 S (Mr approximately 120,000), is isoelectric at pH 4.6, and contains histones H3 and/or H4 tightly bound to one polypeptide of a pair of very acidic polypeptides, designated N1 and N2 (Kleinschmidt, J. A., and Franke, W. W. (1982) Cell 29, 799-809). This complex can be selectively immunoprecipitated by guinea pig antibodies against purified protein N1/N2. In contrast, a larger complex of 7 S contains four histones and nucleoplasmin (the purified protein exists as a pentamer of a polypeptide of Mr approximately 30,000), is isoelectric over the pH range of 5-7, and can be immunoprecipitated by nucleoplasmin antibodies. Its relative molecular weight of 130,000-170,000, as determined by gel filtration, sucrose density gradient centrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked complexes, excludes the association of a histone octamer with nucleoplasmin. In addition to histones H2A and H2B, two histones (designated H3 and H4) which are similar in their electrophoretic mobilities to histones H3 and H4 but have lower isoelectric pH values are enriched in immuno-precipitates obtained with nucleoplasmin antibodies. Cross-linking of complexes present in intact nuclei, using 1% formaldehyde at near-physiological ionic strength and pH, indicates the coexistence of these two soluble histone complexes in the living cell. In chromatin assembly experiments using SV 40 DNA, both histone fractions are able to transfer histones to DNA, resulting in an increase of DNA superhelicity and the formation of beaded nucleoprotein complexes of nucleosome-like morphology. The common principle governing both types of complexes, i.e. the association of one or two histone molecules with a karyophilic large acidic histone-binding protein is emphasized. We discuss the possible role of these complexes in storing histones utilized in chromatin assembly during early amphibian embryogenesis as well as the possible existence of similar complexes, albeit at lower concentrations, in somatic cells.     

The histone-fold complex MHF is remodeled by FANCM to recognize branched DNA and protect genome stability

Histone-fold proteins typically assemble in multiprotein complexes to bind duplex DNA. However, one histone-fold complex, MHF, associates with Fanconi anemia (FA) protein FANCM to form a branched DNA remodeling complex that senses and repairs stalled replication forks and activates FA DNA damage response network. How the FANCM-MHF complex recognizes branched DNA is unclear. Here, we solved the crystal structure of MHF and its complex with the MHF-interaction domain (referred to as MID) of FANCM, and performed structure-guided mutagenesis. We found that the MID-MHF complex consists of one histone H3-H4-like MHF heterotetramer wrapped by a single polypeptide of MID. We identified a zinc atom-liganding structure at the central interface between MID and MHF that is critical for stabilization of the complex. Notably, the DNA-binding surface of MHF was altered by MID in both electrostatic charges and allosteric conformation. This leads to a switch in the DNA-binding preference — from duplex DNA by MHF alone, to branched DNA by the MID-MHF complex. Mutations that disrupt either the composite DNA-binding surface or the protein-protein interface of the MID-MHF complex impaired activation of the FA network and genome stability. Our data provide the structural basis of how FANCM and MHF work together to recognize branched DNA, and suggest a novel mechanism by which histone-fold complexes can be remodeled by their partners to bind special DNA structures generated during DNA metabolism.     

Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA

The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn²⁺ and readily competes with zinc finger peptides for Zn²⁺ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn₃-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn₃-Sp1 under the conditions of the assay that permit some Zn₃-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn₃-Sp1 by GC1, EDTA readily reacted with Zn₃-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn₃-Sp1 failed to compete for zinc bound in the Zn₃-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn₃-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.     

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.     

Binding of low mobility group protein from rat liver chromatin with histones studied by chemical cross-linking

The protein of molecular weight about 160 kD (designated LMG160) was isolated from purified low mobility group chromatin proteins. Polyclonal antibody directed against the LMG160 protein in mouse was raised. The specificity of the antibody was determined with the use of ELISA. Using chemical cross-linking procedure followed by immunoprecipitation with the antiLMG160 antibody complex formation with chromatin proteins was demonstrated. Among the proteins that form complexes with LMG160, histones H3, H2A, and H4 were identified (Western blotting technique).     

Nucleosome assembly in vitro: separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes.

High speed supernatants of Xenopus laevis oocyte nuclei efficiently assemble DNA into nucleosomes in vitro under physiological salt conditions. The assembly activity cofractionates with two histone complexes composed of the acidic protein N1/N2 in complex with histones H3 and H4, and nucleoplasmin in complex with histones H2B and H2A. Both histone complexes have been purified and their nucleosome assembly activities have been analysed separately and in combination. While the histones from the N1/N2 complexes are efficiently transferred to DNA and induce supercoils into relaxed circular plasmid DNA, the nucleoplasmin complexes show no supercoil induction, but can also transfer their histones to DNA. In combination, the complexes act synergistically in supercoil induction thereby increasing the velocity and the number of supercoils induced. Electron microscopic analysis of the reaction products shows fully packaged nucleoprotein structures with the typical nucleosomal appearance resulting in a compaction ratio of 2.8 under low ionic strength conditions. The high mobility group protein HMG-1, which is also present in the soluble nuclear homogenate from X. laevis oocytes, is not required for nucleosome core assembly. Fractionation experiments show that the synergistic effect in the supercoiling reaction can be exerted by histones H3 and H4 bound to DNA and the nucleoplasmin complexes alone. This indicates that it is not the synchronous action of both complexes which is required for nucleosome assembly, but that their cooperative action can be resolved into two steps: deposition of H3 and H4 from the N1/N2 complexes onto the DNA and completion of nucleosome core formation by addition of H2B and H2A from the nucleoplasmin complexes.     

Isolation,characterization, and structure of the folded interphase genome of Drosophila melanogaster

The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% Nonidet P40. The nonviscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranucleosome histones: H2A, H2B, H3, and H4).The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs—that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26°C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres.Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA.The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA-DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360° left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.