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PubMed1983

Protein-primed initiation of phage phi 29 DNA replication.

We recently reported the development of an in vitro replication system for bacteriophage phi 29 DNA. We have used this system for the isolation of replication activity associated with gene 3 protein (terminal protein) from phi 29-infected Bacillus subtilis cells. We utilized two assay systems: (i) DNA replication dependent on phi 29 DNA with the 5' end covalently linked to terminal protein (DNA-protein) and (ii) the formation of complex between the terminal protein and dAMP. The DNA-replication and the complex-forming activities were purified together through all steps. The complex of terminal protein and dAMP formed in the purified fraction was shown to serve as an effective primer for successive chain elongation in the presence of dNTPs by a pulse-chase experiment. The protein fraction purified from cells infected with a temperature-sensitive phi 29 mutant in gene 3 was thermolabile compared to the wild-type activity in the assay system for complex formation. This shows that the purified fraction having replication activity includes the gene 3 product of phi 29. Both the DNA replication and the complex formation activities are highly specific for phi 29 DNA-protein as template. The product analysis of elongated DNA revealed that the replication starts at both termini of the phi 29 genome. These results are consistent with the basic elements of the protein-priming model for the initiation of linear DNA synthesis.

Bacillus subtilis↗
PubMed1980

Spin-label study of histone H1-DNA interaction. Comparative properties of the central part of the molecule and the N and C-amino tails.

Covalent spin labelling of lysine and arginine residues in histone H1 under specified conditions allows the study of histone-DNA interaction. When the labelled histone is free in solution, the electron spin resonance spectrum is characteristic of a label moving in a viscous medium while the label becomes fully immobilized upon the interaction of histone and DNA. This property is used to study histone-DNA interactions under various conditions of ionic strength and thermal denaturation. On the other hand, spin label probes in the globular part of the molecule shows no strong immobilization upon binding to linear DNA, suggesting a special mechanism for the binding of histone H1 to chromatin.

Animals↗
PubMed1987

Toxicity of 3-aminobenzamide to Chinese hamster cells containing 5-hydroxymethyluracil in their DNA.

V79 cells incorporated 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) into their DNA linearly over a wide range of concentrations and time. Cells grew normally when 0.03% of thymidine residues were replaced with HmdUrd. At this level of substitution, 5-hydroxymethyluracil (HmUra) was removed from DNA at a rate of 30-40%/24 h. Concentrations of HmdUrd in the growth medium which produced higher levels of substitution reduced survival and caused cells to delay their transit through S phase. However, the treatment of HmdUrd-containing cells with 3-aminobenzamide caused extensive cell death. At levels of HmdUrd substitution compatible with near 90% survival, the addition of 3-aminobenzamide, an inhibitor of poly (adenosine diphosphoribose) synthesis, killed over 90% of the cells. This toxicity was not due to inhibition of the removal of HmUra from DNA. Cells killed by this combination of agents arrested in the G2 phase of the cell cycle. We conclude that the toxicity of HmdUrd resulted primarily from the repair of the HmUra residue in DNA and not from any intrinsic toxicity of the HmUra residue itself. We also conclude that the cytotoxicity of 3-aminobenzamide resulted from interference with the completion of DNA repair following base (HmUra) excision. Since HmUra is also formed in DNA through the action of ionizing radiation, it may be among the components of radiation-induced DNA damage which sensitizes cells to 3-aminobenzamide.

Animals↗
PubMed1992

Estimating genomic distance from DNA sequence location in cell nuclei by a random walk model.

The folding of chromatin in interphase cell nuclei was studied by fluorescent in situ sequences chromatin according to a random walk model. This model provides the basis for calculating the spacing of sequences along the linear DNA molecule from interphase distance measurements. An interphase mapping strategy based on this model was tested with 13 probes from a 4-megabase pair (Mbp) region of chromosome 4 containing the Huntington disease locus. The results confirmed the locations of the probes and showed that the remaining gap in the published maps of this region is negligible in size. Interphase distance measurements should facilitate construction of chromosome maps with an average marker density of one per 100 kbp, approximately ten times greater than that achieved by hybridization to metaphase chromosome. achieved by hybridization to metaphase chromosomes.

Cell Nucleus↗
PubMed2002

Generation and characterization of transgenic mice expressing cobra venom factor.

Cobra venom factor (CVF), the anticomplementary protein in cobra venom, activates the alternative complement pathway, eventually leading to complement consumption. Here, we describe the development of a transgenic mouse model for CVF. We generated a DNA construct containing the full-length cDNA for single-chain pre-pro-CVF. Expression of CVF was controlled by the alpha(1)-antitrypsin promoter to achieve liver-specific expression. Linearized DNA was microinjected into murine ovary cells (strain CD(2)F(1) (BALB/cxDBA/2J)) and the newborn mice were analyzed for stable integration of CVF DNA. After establishing the transgene, mice were propagated in a BALB/c background. The CVF mRNA was detected in the liver and, in some animals, in the kidney. CVF protein was detected in small amounts in the serum. Serum complement hemolytic activity in CVF-transgenic mice was virtually absent. The concentration of plasma C3 was significantly reduced. The CVF-transgenic animals show no unusual phenotype. They provide an animal model to study the effect of long-term complement depletion by continued activation, as well as the role of complement in host immune response and pathogenesis of disease.

Animals↗
PubMed2002

Dominant effect of protein charge rather than protein shape in apparent DNA bending by engineered bZIP domains.

We are interested in how asymmetric charge neutralization of DNA by proteins results in DNA bending. We have previously reported electrophoretic phasing experiments utilizing homodimer peptides derived from GCN4, a yeast basic zipper (bZIP) transcription factor. Here we report the results of experiments that examine the importance of peptide sequence context in DNA bending and test the hypothesis that peptide structural asymmetry causes electrophoretic anomalies in the absence of DNA bending. We prepared two new series of bZIP peptides that differed dramatically in overall size, structure, and peptide sequence near the DNA. The magnitude of apparent DNA bending is independent of the structure of the protein. This result reduces the concern that bZIP protein structure causes electrophoretic anomalies in the absence of DNA bending. In all cases, both the magnitude and direction of the apparent DNA bend angle are strongly dependent on the local peptide charge. We attempted to validate independently our results with a minicircle competition binding assay. Binding preferences of severalfold for properly phased circular versus linear DNA templates were predicted. However, no binding preferences were observed. We propose that the minicircle binding assay may be intrinsically insensitive to DNA bending or flexibility induced by the bZIP peptides studied, and we provide a unifying explanation for the discrepancies between the cyclization and electrophoretic experiments.

Amino Acid Sequence↗
PubMed1984

Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome.

The human T-cell leukemia (lymphotropic) virus type III (HTLV-III) appears to be central to the causation of the acquired immune deficiency syndrome (AIDS). Two full-length integrated proviral DNA forms of HTLV-III have now been cloned and analyzed, and DNA sequences of the virus in cell lines and fresh tissues from patients with AIDS or AIDS-related complex (ARC) have been characterized. The results revealed that (i) HTLV-III is an exogenous human retrovirus, approximately 10 kilobases in length, that lacks nucleic acid sequences derived from normal human DNA; (ii) HTLV-III, unlike HTLV types I and II, shows substantial diversity in its genomic restriction enzyme cleavage pattern; (iii) HTLV-III persists in substantial amounts in cells as unintegrated linear DNA, an uncommon property that has been linked to the cytopathic effects of certain animal retroviruses; and (iv) HTLV-III viral DNA can be detected in low levels in fresh (primary) lymphoid tissue of a minority of patients with AIDS or ARC but appears not to be present in Kaposi's sarcoma tissue. These findings have important implications concerning the biological properties of HTLV-III and the pathophysiology of AIDS and Kaposi's sarcoma.

Acquired Immunodeficiency Syndrome↗
PubMed1992

DNA cassettes containing the origin of transfer (oriT) of two broad-host-range transfer systems.

Plasmid constructs are described that carry retrievable DNA cassettes containing the origin of transfer region (oriT) from two broad-host-range plasmids. Restriction of these high copy number plasmids with any one of a variety of enzymes yields a linear DNA fragment of convenient size containing the oriT region of either pCUI or RK2. This DNA can be ligated into any vector or recombinant plasmid containing a compatible enzyme site and can be easily identified by size on an agarose gel. Any plasmid can therefore be mobilized using a number of helper strains or conjugative plasmids derived from the parental plasmids. In addition, the cassettes can be used for a variety of genetic manipulations including "selectable" linker mutagenesis.

Cloning, Molecular↗
PubMed1998

Differential effects of heavy metals on the binding of Xenopus upstream binding factor(xUBF) to DNA.

Xenopus upstream binding factor (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. Among these DNA-binding motifs, HMG box I is essential for promoting RNA polymerase I-dependent rRNA gene transcription. Gel shift assay indicated that the binding of recombinant HMG box I to a 136-bp linear DNA probe was significantly inhibited by Cd2+ at 1 microM. The formation of larger protein-DNA complexes was particularly sensitive to Cd2+. The interaction between HMG box I and DNA was completely inhibited by 10 microM of Cd2+, yet this interaction was not inhibited by the same concentration of Ca2+. Hg2+ at 0.1 microM began to cause abnormal band shifting, and protein-DNA bands disappeared to the wells of a polyacrylamide gel in the presence of 10 microM of Hg2+, reflecting that a drastic change in the conformation of HMG box I-DNA had occurred. The binding of HMG box I to DNA was slightly disturbed by As3+ at 1 microM and was significantly affected at 10 microM. Our results suggest that inhibition of the normal binding of UBF to its target DNA may be one of the mechanisms of heavy metal-induced inhibition of RNA synthesis.

Animals↗
PubMed1997

Two vectors for the insertion of mammalian selectable genes into yeast artificial chromosome cloned DNA.

The introduction of cloned DNA into mammalian cells allows functional testing of genes contained on the fragments. In many cases, the exogenous DNA introduced into mammalian cells requires selectable genes that mark the presence of input DNA. Two new vectors, carrying mammalian selectable markers encoding for either neomycin-resistance (neo) or histidinol-resistance (hol), have been constructed for targeted integration to specific single-copy sites within yeast artificial chromosome (YAC) insert DNA. The integration cassettes comprise a single selectable yeast gene adjacent to a mammalian selectable gene, either LEU2 with neo or HIS3 with hol. Modification of the YAC occurs in yeast by transfection with linear DNA containing YAC-specific, unique, recombinogenic ends, thereby ensuring co-integration of the markers. Analysis of modified YACs confirms that both vectors correctly integrate into the targeted unique sites. The precise localization of selectable marker genes in the cloned DNA ensures the integrity of the genomic fragments during functional testing. Placement of mammalian selectable markers within the YAC insert DNA should allow for YAC-based gene targeting experiments in a variety of mammalian cell lines.

3-Isopropylmalate Dehydrogenase↗
PubMed1976

The effects of deoxyribonucleic acid secondary structure on tertiary structure.

The secondary structure of supercoiled DNA was varied by changes in ionic strength. For I = 0.075-0.4 the structure remained in the previously established branched form with only minor alterations in molecular dimensions. In 4M-NaCl, which induces linear DNA to change its secondary structure to the C structure and brings about an increase in the superhelix density of the molecule, no extra branches were observed on the molecules. The limiting factors that dictate supercoil structure seem to be the number and position of potential branch points and the proximity with which the two intertwining DNA strands can approach each other on the arms of the branches. This value is close to 10nm under the conditions described, and is 14-15nm at I = 0.2. It is suggested that such values should be borne in mind when models of chromosome structure are being constructed.

Bacteriophages↗
PubMed2006

Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair.

The function of human XPA protein, a key subunit of the nucleotide excision repair pathway, has been examined with site-directed substitutions in its putative DNA-binding cleft. After screening for repair activity in a host-cell reactivation assay, we analyzed mutants by comparing their affinities for different substrate architectures, including DNA junctions that provide a surrogate for distorted reaction intermediates, and by testing their ability to recruit the downstream endonuclease partner. Normal repair proficiency was retained when XPA mutations abolished only the simple interaction with linear DNA molecules. By contrast, results from a K141E K179E double mutant revealed that excision is crucially dependent on the assembly of XPA protein with a sharp bending angle in the DNA substrate. These findings show how an increased deformability of damaged sites, leading to helical kinks recognized by XPA, contributes to target selectivity in DNA repair.

Cross-Linking Reagents↗
PubMed1998

High-speed separation of linear and supercoiled DNA by capillary electrophoresis. Buffer, entangling polymer, and electric field effects.

Capillary electrophoresis in dilute and semidilute (slightly entangled) hydroxyethyl cellulose (HEC) is shown to separate linear double-stranded DNA (ds-DNA) and supercoiled plasmid DNA in the size range 1-16 thousand base pairs in 3 min. The mobilities of linear ds-DNA fragments are stronger functions of electric field strength and buffer concentration than the mobilities of supercoiled plasmids. The effects of HEC concentration and molecular weight are similar for both forms of DNA. The behavioral differences, which are attributed to the greater stiffness of the plasmids, can be used to define conditions that maximize resolution of supercoiled and linear ds-DNA of the same or similar number of base pairs.

Buffers↗
PubMed1990

Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures.

Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3'-exonuclease and 5'-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.

Base Sequence↗
PubMed1993

Transcription by SP6 RNA polymerase exhibits an ATP dependence that is influenced by promoter topology.

Transcription of linearized DNA templates by SP6 RNA polymerase requires a higher concentration of ATP than of the other three nucleotides. This requirement is not shared by T7 RNA polymerase. The ATP requirement is partially relieved when the SP6 template is supercoiled but not when it is relaxed circular DNA. The effect of supercoiling is eliminated by replacement of the A.T rich sequence downstream from the SP6 promoter with a G.C rich sequence. Examination of the reaction products indicates that the ATP dependence of transcription from a linear template is not due to an ATPase activity or to the premature termination of transcription at low ATP concentration. These data suggest that the initiation of transcription by SP6 RNA polymerase requires partial denaturation of the template in the promoter-proximal region, and that this requirement can be satisfied by negative supercoiling or by increasing the ATP concentration. ATP also reduces, but does not eliminate, the abortive transcription that leads to the production of short, prematurely terminated transcripts by SP6 polymerase from supercoiled templates.

Adenosine Triphosphate↗
PubMed1987

Identification of intrinsic termination sites in vitro for RNA polymerase II within eukaryotic gene sequences.

We have identified and mapped several DNA sequences within a human histone gene (H3.3) at which in-vitro transcription by highly purified RNA polymerase II is efficiently terminated. Since transcription in our system involves only RNA polymerase II acting on a linear DNA template, these sequences contain "intrinsic" termination signals recognized by the polymerase protein itself. The existence of such signals within a gene suggests that efficient antitermination systems probably exist for mammalian transcription units. Alternatively, there could be a high frequency of premature transcription termination, or "polarity" for genes such as H3.3. Intrinsic transcription termination sites in H3.3 are located in sequences of consecutive thymidylate residues (5 to 8 nucleotides) on the non-transcribed DNA strand (T-runs), from which it is likely that such T-runs are elements of the intrinsic termination signal for RNA polymerase II. However, transcription proceeds without significant termination through many similar T-runs, from which it follows that these intrinsic termination signals include other elements. Since transcription is also terminated efficiently at these sites when the transcript remains bound along its full length as a DNA-RNA hybrid, it is unlikely that formation of specific RNA secondary structures in the transcript is a general element of the intrinsic termination signal. Although DNA sequences downstream from the coding portion of the mouse beta-globin gene have been implicated as sites of transcription termination in vivo, these regions do not contain strong intrinsic termination signals, and transcription in vitro proceeds through these regions almost undiminished. Transcriptional termination in this region in vivo may depend on the presence of termination factors or other intracellular elements, and there may be multiple classes of DNA signals that control eukaryotic termination.

Animals↗
PubMed2006

Purification and biochemical characterization of ataxia-telangiectasia mutated and Mre11/Rad50/Nbs1.

Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is activated by DNA double strand breaks to phosphorylate many cellular proteins involved in cell cycle regulation and DNA repair. We have shown previously that the activation of ATM can be reconstituted in an in vitro system using recombinant human ATM. In this system, ATM activity is dependent on the Mre11/Rad50/Nbs1 (MRN) complex and linear DNA, similar to requirements observed in human cells. This chapter describes methods used for the overexpression and purification of human ATM and MRN, as well as a protocol for in vitro kinase assays.

Acid Anhydride Hydrolases↗
PubMed1979

Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro.

Soluble extracts of adenovirus-infected HeLa cell nuclei support DNA replication on exogenous adenovirus DNA templates. Conditions of synthesis using both wild-type and temperature-sensitive extracts have been defined. Nuclear extracts prepared from cells permissively infected with the adenovirus mutant H5ts125 expressed the temperature-sensitive phenotype and could be inactivated at 37 degrees C in vitro. These extracts were completely complemented by the addition of wild-type adenovirus DNA binding protein but not by H5ts125 DNA binding protein. Enhancement by binding protein in the mutant extracts represents replication, as demonstrated by the production of full-sized products and orderly chain elongation originating, as in vivo, at both ends of the linear DNA. Replicative synthesis required the 5'-terminal protein bound covalently to template DNA and could be inhibited by denaturation of this 55,000-dalton protein. Various inhibitors of eukaryotic DNA polymerases, such as aphidicolin and 2',3'-dideoxythymidine triphosphate, inhibited replication of exogenous adenovirus templates in this system as they do in previously reported systems that only elongate endogenous replicating intermediates.

Adenoviruses, Human↗