Formation of sheared G:A base pairs in an RNA duplex modelled after ribozymes, as revealed by NMR.

The thermal stability and structure of an RNA duplex, r(GGACGAGUCC)2, the base sequence of which was modelled after both a hammerhead ribozyme and a lead ribozyme, were studied by CD and NMR. We previously demonstrated that the corresponding DNA duplex, d(GGACGAGTCC)2, formed unique 'sheared' G:A base pairs, where an amino proton, instead of an imino proton, of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head' (Nucleic Acids Res. (1993) 21, 5418-5424). CD melting profiles showed that the RNA duplex is thermally more stable than the corresponding DNA duplex. NMR studies revealed that sheared G:A base pairs are formed in the RNA duplex, too, although the overall structure of the RNA is the A form, which differs from the B form taken on by the corresponding DNA. A model building study confirmed that sheared G:A base pairs can be accommodated in the double helical structure of the A form. A difference between the RNA and DNA duplexes in the stacking interaction involving G:A mismatch bases is also suggested. The demonstration that sheared G:A base pairs can be formed not only in DNA but also in RNA suggests that this base pairing plays an important role regarding the RNA structure.     

Chromatin assembly in isolated mammalian nuclei.

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.     

Comparative Studies of the Thermodynamic Stabilities Between Sheared A:G and Watson-Crick A:U(T) Base Pairs in RNA and DNA

Abstract

Thermodynamic parameters for duplex formation were determined from CD melting curves for r(GGACGAGUCC)₂ and d(GGACGAGTCC)₂, both of which form two consecutive ‘sheared’ A:G base pairs at the center [Katahira et al. (1993) Nucleic Acids Res. 21, 5418–5424; Katahira et al., (1994) Nucleic Acids Res. 22, 2752–27591. The parameters were determined also for r(GGACUAGUCC)₂ and d(GGACTAGTCC)₂, where the A:G mismatches are replaced by Watson-Crick A:U(T) base pairs. Thermodynamic properties for duplex formation are compared between the sheared and the Watson-Crick base pairs, and between RNA and DNA. Difference in the thermodynamic stability is analyzed and discussed in terms of enthalpy and entropy changes. The characteristic features in CD spectra of RNA and DNA containing the sheared A:G base pairs are also reported.

     

Mapping the Mutation Site of an Autographa californica Nuclear Polyhedrosis Virus Polyhedron Morphology Mutant

A polyhedron morphology mutant of Autographa californica nuclear polyhedrosis virus, designated M5, was compared with wild-type virus by genotypic analysis with EcoRI, BamHI, HindIII, SstI, and SmaI restriction endonucleases. M5 DNA revealed several alterations relative to the wild-type pattern: (i) EcoRI fragment I was 400 base pairs larger; (ii) BamHI fragment F was missing; (iii) HindIII fragment F was 400 base pairs larger; (iv) an extra restriction fragment was obtained with both HindIII and SmaI; and (v) SstI fragment G was 400 base pairs larger. M5 virions contained two size classes of circular DNA, one of 100% of the wild type and one of about 58% of the wild-type molecule. A revertant of M5, designated M5R, was isolated on the basis of polyhedron morphology. The genome of M5R contained the insertion of DNA in EcoRI fragment I and in HindIII fragment F, but was similar to the wild type in its other restriction fragment patterns. M5-infected cell cultures synthesized a polyhedrin polypeptide smaller in size than the wild type or M5R.     

Unusual DNA duplex and hairpin motifs

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G·A base pairs are separated by one or two pairs of purine·purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G·C or A·T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G·A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G·A, A·A, A·C or sheared-like G_anti·C_syn base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.     

New map of bacteriophage lambda DNA.

A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes. Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to DNA molecules of known sequence. The data were subjected to least-squares analysis to assign map coordinates. In no case did a fragment size predicted from the map differ from the measurement of the fragment by more than +/- 5%. This low error rate was consistent in all size ranges of fragments. The total length of lambda was calculated as 49,133 nucleotide pairs. This probably is accurate to within 500 base pairs.     

Relation between the transforming activity of a marker and its proximity to the end of the DNA particle

Summary Tranforming pneumococcal DNA is inactivated by treatment with restriction enzymes. For mutations belonging to the same locus (amiA locus), the extent of inactivation depends strongly upon the mutations and the enzymes. Two EcoRI and one BamHI restriction sites have been located within the amiA locus. After treatment of donor DNA with either one of these enzymes, the lowest transforming activity is observed for mutations that map near restriction sites. This effect of proximity to the nearest end of the DNA fragment extends over a distance of 1,400 nucleotides. The curve of transforming activity versus DNA size obtained with endonuclease-generated DNA fragments is very similar to that obtained previously with mechanically sheared DNA. Both curves show a striking slope change for donor DNA size around 2,700 base pairs, i.e. twice the length found for the extent of the end effect. We suggest that for donor DNA fragments larger than 2,700 base pairs the transforming activity depends mainly upon the size of donor whereas for donor DNA fragments shorter than 2,700 base pairs both a size-dependent phenomenon and the end effect contribute to reduce drastically the transforming activity.     

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.     

Interaction of Hoechst 33258 with repeating synthetic DNA polymers and natural DNA

Fluorescence, circular dichroism and sedimentation through cesium chloride gradient techniques were performed to study the physical properties of the binding of the bisbenzimidazole dye Hoechst 33258 (H33258) to natural DNAs and synthetic polynucleotides of defined repeating units. These studies show that Hoechst 33258 exhibits at least two modes of interaction with duplex DNA: (1) a strong base pair specific mode which requires at least 4 consecutive AT base pairs and (2) a weaker mode of binding which is significantly reduced in the presence of high salt (0.4 M NaCl) and exhibits no apparent base specificity. The H33258 binding was found to be sensitive to the substitutions in the minor groove elements of a series of synthetic polynucleotides supporting the model of H33258 binding in the minor groove of the DNA with AT rich sequences. Similar mode of binding was predicted in natural DNAs by methylation of dye-DNA complexes. Footprint analysis of the complex of dye to a pBR322 fragment also supports that a minimum of 4 consecutive AT base pairs are required for H33258 binding to DNA.     

Activity of Deoxyribonucleic Acid Fragments of Defined Size in Bacillus subtilis Transformation

The transforming activity of Bacillus subtilis deoxyribonucleic acid (DNA) that had been sheared and purified with respect to size by sucrose gradient sedimentation is given as a function of the DNA molecular weight. It is shown (i) that fragments of median molecular weight 1.2 x 10(6) have finite activity (10(-4)), (ii) that the shape of the activity-versus-molecular weight function is qualitatively similar to that observed previously for Diplococcus pneumoniae, and (iii) that this shape precludes interpretation in terms of critical size models.     

Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

Defective Bacteriophage PBSH in Bacillus subtilis: I. Induction, Purification, and Physical Properties of the Bacteriophage and Its Deoxyribonucleic Acid

PBSH, a defective phage of Bacillus subtilis strain 168, is described. Conditions are given for optimal induction of the prophage with mitomycin C. After a latent period of 90 min, cells were lysed and phage-like particles were released with a burst size of approximately 100 to 400 phage per bacterium. Since no known host supports phage replication after infection, burst size was determined by electron microscope count. Purification procedures and criteria for purity are described. The molecular weight of deoxyribonucleic acid (DNA) extracted from PBSH was estimated by length measurement and sedimentation. No circular DNA molecules were found by either technique. PBSH DNA molecules are linear, double-stranded, and of homogeneous molecular weight, about 12 x 10(6) daltons. There is no evidence for single-strand breaks. The majority of PBSH DNA molecules show a sedimentation behavior dependent on ionic strength. It is inferred that most of the DNA molecules are less hydrodynamically rigid than native DNA having a similar average base composition and molecular weight. Possible reasons for the sedimentation behavior are discussed.     

Fragmentation of chromatin by endogenous nucleases, accumulation of the subnucleosomal fragments with high electrophoretic mobility]

Digestion of chromatin by endogenous nucleases to nucleosomes (140-160 base pairs of DNA) is accompanied by the accumulation of subnucleosomal DNP particles with high electrophoretic mobility (20-40 base pairs of DNA). All histones associate with the 140-160 base pairs fragment. The production of subnucleosomal DNP particles does not correlate with the degradation of histone H1 and the appearance of nucleosomes lacking histone H1. Degradation of the protein in this fragment is accompanied by the appearance of free DNA. The data obtained are in agreement with the hypothesis on the origin of subnucleosomes from the nucleosomal locus preferentially associated with the non-histone proteins and on the autonomy of these loci and of the loci associated with histone H1 in the nucleosome.     

Chromomycin, mithramycin, and olivomycin binding sites on heterogeneous deoxyribonucleic acid. Footprinting with (methidiumpropyl-EDTA)iron(II)

The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE x Fe(II)] DNA cleavage inhibition pattern technique. Two DNA restriction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with 32P at the 3' end. MPE x Fe(II) was allowed to partially cleave the restriction fragment preequilibrated with either chromomycin, mithramycin, or olivomycin in the presence of Mg2+. The preferred binding sites for chromomycin, mithramycin, and olivomycin in the presence of Mg2+ appear to be a minimum of 3 base pairs in size containing at least 2 contiguous dG x dC base pairs. Many binding sites are similar for the three antibiotics; chromomycin and olivomycin binding sites are nearly identical. The number of sites protected from MPE x Fe(II) cleavage increases as the concentration of drug is raised. For chromomycin/Mg2+, the preferred sites on the 70 base pairs of DNA examined are (in decreasing affinity) 3'-GGG, CGA greater than CCG, GCC greater than CGA, CCT greater than CTG-5'. The sequence 3'-CGA-5' has different affinities, indicating the importance of either flanking sequences or a nearly bound drug.     

Characterization of a highly repetitive family of DNA sequences in the mouse.

A large proportion (0.5-1%) of total mouse DNA is cleaved by Bam HI into fragments whose size is about 500 base pairs. A cloned member of this repetitive family of DNA sequences (BAM5 family) was sequenced by the dideoxy chain termination procedure and shown to contain 507 base pairs. The sequence exhibited no unusual or remarkable features. Repetitive sequences complementary to the cloned BAM5 fragment were found in rat DNA, but not in feline or human DNA. Restriction mapping suggested that many BAM5 sequences were components of much larger repetitive DNAs which were scattered throughout the mouse genome. The BAM5 sequences within the larger repetitive DNAs did not appear to be arranged tandemly or as members of scrambled tandem repeats. RNA homologous to the cloned BAM5 sequence was detected in cultured mouse cells, but not in cultured rat cells.     

Sequence organization of the soybean genome

The total complexity of one constituent soybean (Glycine max) genome is estimated to be 1.29 . 10(9) nucleotide pairs, as determined by analysis of the reassociation kinetics of sheared (0.47 kilobase) DNA. Single copy sequences are estimated to represent from 53 to 64% of the genome by analysis of hydroxyapatite binding of repetitive DNA as a function of fragment length. From 65 to 70% of these single copy sequences have a short period interspersion with 1.11--1.36 kilobase lengths alternating with 0.3--0.4 kilobase repetitive sequence elements. The repetitive sequences of soybean DNA are interspersed both among themselves and among single copy regions of the genome.     

Isolation and characterization of chromatin subfractions from rat liver

Rat-liver chromatin was digested with micrococcal nuclease at low ionic strength in the presence of a low concentration of CaCl2. The nuclease digest was successfully separated into three fractions, P1, P2, and P3, by gel filtration on a column of Sepharose 2B. P1 fraction was shown to be a mixture of long fragments of partially digested chromatin by the sedimentation profile or by electrophoresis of DNA. P2 fraction contained four histones H2A, H2B, H3, and H4 in almost equal amounts, together with nonhistone protein of low molecular weight. The DNA was composed of three or four fragments less than 300 base pairs long. From the Kav value of the P2 fraction, the average size was estimated to be about 240 base pairs. On analytical ultracentrifugation, this fraction exhibited a monophasic boundary and a sedimentation value of 13.7S. P3 fraction contained nonhistone proteins which showed a molecular weight larger than that of H1 histone. The size of DNA was estimated to be less than 50 base pairs from the Kav value. Based on these results, the P2 fraction was concluded to consist of nucleosome monomer enriched in nonhistone proteins. The P3 fraction is presumably the nuclease-sensitive or internucleosome portion, which contains small amounts of nonhistone proteins.     

Cloning of randomly sheared DNA fragments from a phi 105 lysogen of Bacillus subtilis identification of prophage-containing clones

A gene bank for Bacillus subtilis has been developed by cloning randomly sheared DNA fragments of a B. subtilis (phi 105) lysogen DNA in Escherichia coli employing the pMB9 plasmid vector. The DNA was inserted by the oligo(dA)-oligo(dT) method, and the average insert size of the cloned DNA was 7 kilobase pairs (kb). Three clones have been identified which carry DNA from the phi 105 prophage. None of these clones contain the phage-chromosome junction.     

Cloning and localization of the repressor gene (c) of the Mu-like transposable phage D108

We have localized the D108 thermosensitive (cts) repressor gene to a region of DNA approx. 600 base pairs (bp) in length by sub-cloning an RsaI restriction endonuclease fragment (bp 200 to bp 802 from the left-end of the D108 genome). We determined that the gene product from this fragment appears to be the same size (19 kDa) as that expressed from clones containing larger fragments of D108 DNA. Results from in vitro gel electrophoresis band-retardation and in vivo immunity assays show that the sub-cloned repressor appears to be fully functional.