Annika C. Arnberg

HELA NUCLEAR-PROTEIN RECOGNIZING DNA TERMINI AND TRANSLOCATING ON DNA FORMING A REGULAR DNA MULTIMERIC PROTEIN COMPLEX

Employing an exonuclease III protection assay we detected a protein in crude HeLa nuclear extracts binding, with apparent sequence specificity, to molecular ends of adenovirus type 2 (Ad2) DNA. This protein, designated nuclear factor IV (NFIV), was purified to homogeneity and was shown to be a hetero-dimer of 72,000 and 84,000 Mr. Binding to terminal Ad2 sequences was strongly enhanced by the presence of either of the sequence-specific DNA-binding proteins nuclear factor I and nuclear factor III. These proteins appeared to function as blockades for translocation of NFIV on DNA, thus producing apparent sequence specificity. In the absence of such a blockade, NFIV moved freely, without energy input, on any double-stranded DNA forming a regular DNA-multimeric protein complex as shown by methidiumpropyl EDTA footprinting and electron microscopy. Binding is completely dependent upon the presence of molecular ends. Evidence was obtained for a two-step mechanism in which termini are recognized by NFIV and used as a starting point for subsequent translocation. The possible functions of the protein in adenovirus DNA replication and in cellular processes requiring DNA termini are discussed.

SOME YEAST MITOCHONDRIAL RNAS ARE CIRCULAR

11S and 18S fractions of yeast mitochondrial RNAs, isolated by electrophoresis through agarose gels, have been found by electron microscopy to contain approximately 50% circular molecules. Circles in the 11S fraction have a contour length of 0.36 +/- 0.02 micron, which is approximately equal to the length of the majority of linear molecules also present. Circles in the 18S fraction have an average length of 0.78 +/- 0.11 micron. The size distribution is broader than for the 11S fraction, and we cannot exclude the possibility that more than one size class may be present. The 11S circular RNA forms circular R loops and RNA-DNA hybrids with DNA fragments of the oxi 3 region of mtDNA, which contains the structural gene for subunit 1 of cytochrome oxidase. As judged from the electron micrographs, the complete RNA participates in hybrid formation and the sequences coding for it appear to be continuous. Both 11S and 18S circles withstand treatment with DNAase and pronase. They are not eliminated by treatment with 1 M glyoxal in 50% formamide for 1 hr at 50 degrees C. We conclude that they are covalently closed. The function of the circular RNAs is unknown. They may be active as mRNAs, storage forms, or arise in a cut-and-splice process which generates mRNAs from longer transcripts.

Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14

SummaryThe genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter favus H4-14 were identified and characterized. The RuBisC/O genes are separated by 11 by and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS. Primer extension and R-loop experiments with RNA isolated from autotrophically grown X. favus H4-14 showed that transcription of cfxL and cfxS initiated 22 by upstream from cfxL and resulted in a mRNA of at least 2.3 kb. DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators. Downstream from cfxS an additional open reading frame was identified with unknown function. Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS. The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.

The presence of DNA molecules with a displacement loop in standard mitochondrial DNA preparations.

Abstract In electron micrographs of standard closed circular duplex mtDNA preparations from chick liver, spread by the protein monolayer technique from solutions containing 40 or 76% formamide, up to 30% of the molecules contained a singled-stranded displacement loop, as depicted in Fig. 1. The average lengths of the loops and of the doublestranded sections between the attachment points were about the same and equivalent to 3.5% of the contour length of the mtDNA circle.

The unusual properties of mtDNA from a “low-density” petite mutant of yeast

Abstract 1. We have purified the mtDNA from the “low-density”, ethidium-induced petite mutant RD1A by repeated CsCl gradient centrifugation and hydroxylaptite chromatography. 2. The density in neutral CsCl of this DNA is 1.671 g/cm 3 and its T m in 0.2 M Na + is 69 °C. Base composition analysis gives A = T and G = C and G+C = 6 mole percent. 3. Quantitative renaturation studies show: (a) Denatured RD1A mtDNA renatures with second-order kinetics and contains no self-complementary single strands detectable by hydroxylapatite chromatography. (b) RD1A mtDNA must consist of a perfect repetition of a sequence of 300 nucleotides or less. (c) RD1A mtDNA is complementary to roughly 0.5 % of wild-type yeast mtDNA. 4. The behaviour of purified RD1A mtDNA on hydroxylapatite suggests that it largely consists of aggregates that are mechanically trapped on the column. Elution required a temperature near the T m of the DNA. 5. RD1A mtDNA sedimented through 3 M alkaline CsCl as heterogeneous linear DNA with a median molecular weight of about 1.7 · 10 6 . 6. In neutral CsCl gradients containing ethidium the bulk of RD1A mtDNA showed a restricted uptake of ethidium; this was abolished by treatment with pancreatic deoxyribonuclease I. 7. Electron micrographs of RD1A mtDNA spread by the protein monolayer technique contained very large networks and an occasional small circle; spreading of the DNA under denaturing conditions confirmed that less than 0.1 weight percent is circular. 8. We propose that most of the isolated RD1A mtDNA is present in large fishnet concatenanes; the unwinding restriction imposed on DNA of which the strands are plaited into a fishnet concatenate explains the restricted ethidium uptake.

An analysis by electron microscopy of intermediates in the replication of linear Tetrahymena mitochondrial DNA

Abstract 1. 1. We have analysed, by electron microscopy, branched forms of Tetrahymena mtDNA, spread in protein monolayers from formamide-containing solutions. These were obtained as fast-sedimenting, pulse-labelled mtDNA from normal cells or from cells grown in ethidium bromide, in which such branched forms accumulate. 2. 2. All branched forms were linear and of the same end to end length as unbranched (linear) Tetrahymena mtDNA. 3. 3. The major type of branched DNA was a duplex with an internal “eye” of variable size; “eyes” were largely duplex but often contained short single-stranded sections in one or both forks, in the latter case usually in the trans position. 4. 4. Other putative replication intermediates found were linear molecules with one single-stranded end, molecules with one “eye” within an “eye” and Y-shaped molecules. 5. 5. The data fit a replication model in which DNA synthesis starts near the middle and proceeds bi-directionally to the ends of the linear molecule. We present a speculative scheme for the conversion of linear DNA with one 3′-single-stranded end into fully duplex molecules, which utilizes our previous finding that this DNA contains a terminal duplication-inversion.

Structure and RNA content of the prosomes

Duck erythroblast, prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo‐)electron microscopy. A molecular weight of ≈ 720,000 ± 50,000, a radius of gyration of 64 ± 2 Å and a hydrodynamic radius of ≈ 86 Å were obtained. Electron micrographs show a hollow cylinder‐like particle with a diameter of 120 Å, n height of 170 Å and a diameter of 40 Å for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.

Structural alterations of double-stranded DNA in complex with the adenovirus DNA-binding protein. Implications for its function in DNA replication.

The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.

DNA synthesis by isolated mitochondria V. Electron microscopy of replicative intermediates

Abstract 1. We have analysed by electron microscopy mtDNA preparations extracted before and after incubation of isolated chick-liver mitochondria with DNA precursors. The only detectable effect of incubation was on molecules with single-stranded gaps co-sedimenting with the open circular form of mtDNA; the average gap size had decreased by 30 % after incubation. 2. Up to 90 % of classifiable molecules of the closed circular duplex fraction of rat-liver mtDNA have displacement loops. mtDNA extracted directly from chick-liver homogenates or from mitochondria isolated in the presence of ethidium bromide, an inhibitor of mtDNA synthesis, contained displacement loops of the same size and at the same frequency as mtDNA from isolated mitochondria. This indicates that D-loops are present in vivo and not formed during the isolation of the mitochondria. 3. These results and our previous biochemical experiments indicate that isolated mitochondria are capable of carrying out at least the first and last stage of the replication scheme proposed for mtDNA by Robberson D. L., Kasamatsu, H. and Vinograd, J. (1972) Proc. Natl. Acad. Sci. U.S. 69, 737–741.

EFFECT OF CONCENTRATION ON THE SUBSEQUENT FATE OF PLASMID DNA IN HUMAN-FIBROBLASTS

SummaryThe physical fate of plasmid DNA after entry into human fibroblasts was studied using Southern hybridisation and electron microscopy. Exposure of the cells (5x105 per well) to pC194 DNA-CaPi, containing 50 μg plasmid DNA, resulted in the occasional formation of interlocked molecules. Exposure to a co-precipitate containing 100 μg pC194 plasmid DNA per well resulted in an increase of interlocked molecules by a factor of 10–20 relative to the number of monomers. In addition, new classes of molecules were observed. After prolonged incubation of the cells exposed to the higher DNA concentration, the plasmid DNA was partly contained in structures with a very low electrophoretic mobility. Upon restriction endonuclease digestion of the re-extracted DNA, a pattern of bands was observed, suggesting the involvement of illegitimate recombination between non-random plasmid DNA sequences in the formation of the new classes of molecules.