On the mechanism of interaction between histone II B1 and DNA and histone II B2 and DNA

A mechanism is suggested at the molecular level whereby histone IIB₂ can act as a cross-link between two (or possibly three) adjacent and parallel strands of DNA double helix some 40 Å apart. Application of Prothero's rule and the Lewis probability functions indicate the probable locations of three a-helices and a number of β-turns. This, coupled with the requirement that the tertiary conformation of the histone be complementary to the DNA molecules and for as many basic groups as possible to bind to phosphate oxygens, allows us to suggest, on the basis of model building using accurate space-filling (CPK) models, a complex conformation that achieves this.A similar process applied to histone IIB₁, whose complete amino acid sequence is also known, shows the location of five probable a-helices, a number of β-turns, and a segment of β-pleated sheet. The basic amino acids are gathered in four groupings. Model building experiments suggest that histone IIB₁ forms a complex strut joining four parallel strands of DNA double helix that form a diamond with diameters 100 and 40 Å. In both these models the purpose and function of a fair proportion of the individual amino acids can be specified.This paper is the third and last of a series in this Journal in which models are presented for the tertiary conformation and function of all five histones of known (in whole or in part) amino acid sequence. This suggests that all five are concerned in packing the long DNA double helix, which may be in a “square helix” form, into the confined space of the chromosome. The hypotheses may be tested by a direct investigation of nucleoprotein in situ to see if these 40, 70, and 100 Å interhelical distances can be detected by biophysical methods.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

Anuran erythrocytes and liver both contain satellite histone Hls

In addition to the usual major Hl sub-fractions, a “satellite” histone Hl^s, similar in amino acid composition and molecular weight to mammalian Hl^o, is present in both liver and erythrocyte nuclei of $\text{Xenopus laevis}$ and $\text{Rana catesbeiana}$ (as in freshwater turtles), but no erythrocyte-specific histone resembling the H5 of birds and some fish was found. Such lysine-rich satellite histones may share functional homologies while displaying tissue-specific variations in composition.     

Effects of DNA Superhelical Stress on the Stability of H2B-Ubiquitylated Nucleosomes

On the nucleosome level, histone posttranslational modifications function mainly as the regulatory signals; in addition, some posttranslational modifications can enhance nucleosome stochastic folding, which is restricted in “canonic” nucleosomes. Recently, it has been shown in vitro that symmetric or asymmetric nucleosome ubiquitylation at H2BK34 (and H2BK120, to a lesser extent) can destabilize one of the nucleosomal H2A–H2B dimers and promote nucleosome conversion to a hexasome particle [Krajewski et al. (2018). Nucleic Acids Res., 46, 7631–7642]. Such lability of H2Bub nucleosomes raises a question of whether they could accommodate transient changes in DNA torsional tensions, which are generated by virtually any process that manipulates DNA strands. Using positively or negatively supercoiled DNA minicircles and homogeneously-modified H2Bub histones, we have found that DNA topology could strongly and selectively affect nucleosome stability depending on its ubiquitylation state (here the term “nucleosome stability” means the nucleosome property to maintain its structural integrity and dynamics characteristic to “canonic” nucleosomes). The results point to a role for H2B ubiquitylation in amplifying or mitigating the effects of a DNA torque on the nucleosome stability and dynamics.     

Exocyclic groups in the minor groove influence the backbone conformation of DNA

Exocyclic groups in the minor groove of DNA modulate the affinity and positioning of nucleic acids to the histone protein. The addition of exocyclic groups decreases the formation of this protein–DNA complex, while their removal increases nucleosome formation. On the other hand, recent theoretical results show a strong correlation between the B_I/B_II phosphate backbone conformation and the hydration of the grooves of the DNA. We performed a simulation of the d(CGCGAATTCGCG)₂ Drew Dickerson dodecamer and one simulation of the d(CGCIAATTCGCG)₂ dodecamer in order to investigate the influence of the exocyclic amino group of guanine. The removal of the amino group introduces a higher intrinsic flexibility to DNA supporting the suggestions that make the enhanced flexibility responsible for the enlarged histone complexation affinity. This effect is attributed to changes in the destacking interactions of both strands of the DNA. The differences in the hydration of the minor groove could be the explanation of this flexibility. The changed hydration of the minor groove also leads to a different B_I/B_II substate pattern. Due to the fact that the histone preferentially builds contacts with the backbone of the DNA, we propose an influence of these B_I/B_II changes on the nucleosome formation process. Thus, we provide an additional explanation for the enhanced affinity to the histone due to removal of exocyclic groups. In terms of B_I/B_II we are also able to explain how minor groove binding ligands could affect the nucleosome assembly without disrupting the structure of DNA.     

Primary organization of the nucleosome core particles sequential arrangement of histones along DNA

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particles has been determined by a new sequencing procedure. The lysine groups of histones were crosslinked to partly depurinated DNA at neutral pH. One strand of DNA was split at the points of crosslinking, thus leaving the 5′-terminal DNA fragments bound to histones. The lengths of these crosslinked DNA fragments were measured to determine the position of histones on one strand of the core DNA from its 5′ end.The results demonstrate that histones are bound to regularly arranged, discrete DNA segments about six nucleotides long. These segments are separated by histone-free gaps about four nucleotides wide located at a distance of about 10n nucleotides from the 5′ end of DNA. The first 20 nucleotides from the 5′ ends of DNA seem to be free of histones. Histones appear to be arranged symmetrically and in a similar way on both DNA strands. Any one histone, being bound predominantly to discrete segments on one or other of the strands, can oscillate at the same time between the two strands across the major DNA groove. Two symmetrical models for the arrangement of two molecules of each core histone on linearized and folded DNA are proposed.     

Interactions of an antitumor platinum compound with deoxyribonucleic acid, histones, L-amino acids, poly-L-amino acids, nucleosides and nucleotides

Interaction of cis-dichloro(dipyridine)platinum(II) (cis-PPC) with calf thymus DNA, calf thymus histone, l-amino acids, poly-l-amino acids, nucleosides, and nucleotides has been evaluated by equilibrium dialysis technics. At least a 28 % decrease in the association of cis-PPC with DNA occurs when the platinum compound is pre-incubated with l-amino acids. The greatest decrease in association is seen upon pre-incubation of the platinum compound with the free amino acids. Glut, Asp, Lys, Arg, and CySH, before the addition of a sack containing a solution of DNA. The low level of association between DNA and the amino acids tends to rule out competition between cis-PPC and amino acids for DNA association sites. cis-PPC was repelled from sacks containing positively charged poly-l-Lys, poly-l-Arg, and calf thymus histone; however, in the presence of poly-l-Glut and poly-l-Asp, cis-PPC associated with these negatively charged polymers to a considerable degree. Enhanced chloride dissociation from cis-PPC was observed in the presence of all of the amino acids and the nucleotides GMP, CMP, UMP, and TMP, but not in the presence of AMP or the nucleosides rG and dG. In the presence of calf thymus histone, the association of cis-PPC with calf thymus DNA was reduced by more than 50% at histone/DNA ratios of 0.8–1.0.These data suggest that cis-PPC or cis-Pt(II) may associate with electron-rich areas of not only nucleic acids and proteins but also with body pools of free nucleotides and amino acids. The presence of positively charged histones shielding DNA strands in vivo suggests that the most probable point of platinum-DNA association would be at de-repressed areas of DNA which are undergoing RNA synthesis. The aquated form of the platinum complex may also associate with acidic proteins which appear to be involved in the positive control of RNA synthesis and, as a result, this interaction may be of pharmacological significance.     

Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus

Polymerases from the Pol-I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol-II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. ‘Rational’ alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild-type Pfu–Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490W), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type. A486, L490 and Y497 are on an α-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this α-helix to a second long helix. None of the four amino acids can contact the dNTP directly. Therefore, the increased selectivity for ddNTPs is likely to arise from two factors: (i) small overall changes in conformation that subtly alter the nucleotide triphosphate binding site such that ddNTPs become favoured; (ii) interference with a conformational change that may be critical both for the polymerisation step and discrimination between different nucleotide triphosphates.     

The multi-domain protein Np95 connects DNA methylation and histone modification

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

Postnatal increase in histone H1a in the rat pancreas

Adult rat pancreas contains an unusually high amount of histone H1a; $\text{H1a}\text{H1}$ ratio = 0.27. Histone H1a is not detectable in embryonic or newborn rat pancreas. This histone begins to appear at about 1 week postnatally and reaches the adult level about 5 weeks after birth. There is an inverse relationship between the appearance of histone H1a and DNA synthesis, as indicated by the nuclear labeling index after [³H]thymidine autoradiography.     

Underwound loops in self-renatured DNA can be diagnostic of inverted duplications and translocated sequences

DNA that contains inverted duplications separated by non-inverted sequences often can form characteristic “underwound loops” when it is denatured and reannealed. An underwound loop is a partially double-stranded, partially denatured segment between the inverted duplications and is produced as follows. During the early stages of the reannealing, intrastrand stem-loop structures form with first-order kinetics when the inverted duplications pair. In a slower second-order reaction, complementary strands (each with a stem-loop) reanneal. The stem-loop structures produce a cruciform in the hybrid. Because of the unpaired sequences in the loop, the cruciform is unstable. It can isomerize to a linear duplex by double-strand exchange of complementary sequences in the stems. This process requires co-ordinated axial rotation of the stems and the flanking duplexes as well as rotation of the loops. If, however, complementary sequences in the loops start to pair, axial rotation is prevented and the stem-loop structures are trapped in a metastable state. The strands of separate, closed rings cannot interwind when they pair. Consequently, the loops observed by electron microscopy have variable patterns of single-stranded denaturation bubbles and duplex segments with both right-handed and left-handed winding.We have used underwound loops to identify a short inverted duplication flanking the γδ recombination sequence of Escherichia coli F factor (isolated on φ80 d₃ilv⁺ transducing phage) and to study DNA from phages Mu and P1 in which the G segments are flanked by inverted duplications. When deproteinized adenovirus-2 DNA was denatured and reannealed, some underwound circles the length of the entire chromosome were observed by electron microscopy. These resulted from the restricted interaction of complementary single-stranded rings generated when pairing of the short inverted terminal duplications closed the ends of single strands. Another type of underwound loop was seen in heteroduplexes containing complementary insertion loops located at different positions in the hybridized strands, such as occurs with P1 cam DNAs. All these underwound structures are similar in appearance to the hybrids formed when topologically separate, complementary single-stranded circles of Colicin E₁ DNA were allowed to anneal.     

Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the french bean Phaseolus vulgaris

The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G₁ by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G₁ slightly before the $\text{G}{}_1\text{S}$ boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G₁, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G₁, resulting in significant G₁-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G₁ before “new” f1 which is synthesized in S phase; and (c) G₁-phase f1 phosphorylation does not require new histone or new DNA synthesis.Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G₁-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G₁-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G₂ to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G₁, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.     

Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts

We have utilized sodium butyrate-treated normal human diploid fibroblasts to study core histone hyperacetylation kinetics. We report a small, distinct population of core histone characterized by a very rapid rate of hyperacetylation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈10–15}\text{min}$ for monoacetylated histone H4) compared to the slower rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈140–200}\text{min}$ for monoacetylated H4) observed for bulk histone. Two rates of core histone deacetylation were also detected and we demonstrated that the rapidly hypermodified histone H4 population was also rapidly deacetylated. The kinetics of histone H4 hyperacetylation and deacetylation in these cells were not significantly altered, regardless of whether cultures were exponentially growing, confluent or arrested in an essentially non-mitotic state.     

Rectification of tandemly repetitious DNA. I. Influences on rate

To account for observed patterns of uniformity and heterogeneity in tandemly repetitious DNA, earlier workers have proposed three intracellular mechanisms: the rolling circle model, gene conversion between unequally aligned sister chromatids, and unequal sister chromatid exchange (SCE). All three are forms of rectification, which is the random replacement of a specific number, β of repeating units by replicates of a group (α in number) of repeating units that are present elsewhere in the segment. For all three forms of rectification: (1) the average level of uniformity, C, in the segment increases with each rectification event by an amount given by $\text{ΔC=}\text{β(α+β)}\text{αn(n?1)}\text{(1?C)}$, in which n is the number of repeating units per segment. In gene conversion and unequal SCE, α = β (2) ΔC is affected by periodicity and clustering. (3) Mutation rate, β and the frequency of rectification events can vary with n so as to offset largely the presence of n(n?1) in the denominator. This allows rectification to operate even with very large numbers of small repeating units.     

Reassociation and dissociation of cytoplasmic membrane-associated DNA

The relationship between nuclear DNA and cytoplasmic membrane-associated DNA, extracted from a human lymphocyte cell line, was examined by DNA-DNA reannealing and by dissociation of renatured molecules. Up to 2% of the total cellular DNA is found in the cytoplasm as cytoplasmic membrane-associated DNA and of this 2%, approximately 70% is comprised of repeated sequences. These sequences are homologous to only about 4% of the repeated sequences of nuclear DNA. The repeat fraction of cytoplasmic membrane-associated DNA consists of sequences which are only moderately repeated. The number of copies in the average “family” could range from about 1500 copies to as few as 25 copies. A small rapidly reannealing portion of cytoplasmic membrane-associated DNA (C₀t < 4 × 10^?3) appears to consist of sequences derived from a single “family”.About 30% of cytoplasmic membrane-associated DNA reassociates slowly with a $\text{C}{}_0\text{t}\text{1}\text{2}$ value of 223 (unique cytoplasmic membrane-associated DNA). This fraction has homology with about 11% of the unique sequences of nuclear DNA. However, unique cytoplasmic membrane-associated DNA comprises only about 0·6% of the total cellular DNA. If it is assumed that each cell has the same amount of cytoplasmic membrane-associated DNA, homology with 11% of the unique sequences of nuclear DNA suggests that different cells may have different unique nucleotide sequences in the cytoplasm.     

The rolling-circle replicative structure of a bacteriophage lambda DNA

Intermediates of λ DNA replication in the second half of the latent period have been isolated and investigated in the electron microscope. The isolated replicative structures were predominantly single-branched “$\text{rolling-circle}$” replicative forms. The long linear tails (concatemers) may be the precursor of mature λ DNA.     

Partial tertiary structure assignment for the acetylcholine receptor on the basis of the hydrophobicity of amino acid sequences and channel location using single group rotation theory

Six transmembrane segments of the α-protein unit of the acetylcholine receptor (AChR) are assigned from the amino acid sequence (M.Toda $\text{et}\text{al}$, Nature 299, 793–797 (1982), two as part of the ion channel using single group rotation (SGR) theory (E.M.Kosower, Abstracts, pp. 52-3, Symposium “Structure and Dynamics of Nucleic Acids and Proteins”, Sept. 1982) and four on the basis of the hydrophobicity of amino acid sequences.