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At least 289 records · Page 16Linked to original sources
PubMed1995

The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence.

The SfiI endonuclease cleaves DNA by a mechanism that differs from other restriction enzymes. While most restriction enzymes are dimeric proteins that interact with a single DNA site, SfiI exists in solution as a tetramer and it appears to interact with two copies of its recognition sequence before it can cleave DNA. Its primary reaction is then to cut both strands at both sites in a concerted process. The two sites can be on either the same or different DNA molecules, so SfiI provides a test system for long-range interactions on DNA. On either supercoiled or linear DNA with two sites separated by 1 kb, the bridging interaction between the sites is an intramolecular event: the majority of the DNA is converted directly to products cleaved at both sites, bypassing intermediates cut at one site. Sites on separate DNA molecules, or two sites on linear DNA several kb apart, engage in an intermolecular interaction prior to cleavage. The interaction between two DNA molecules with one site on each is impeded by supercoiling in both partners but is permitted when one partner is linear: it may require reptation of one DNA through another. SfiI reactions have marked similarities to some of the reactions catalysed by site-specific recombination enzymes.

Base Sequence↗
PubMed1998

A simple mechanism for the avoidance of entanglement during chromosome replication.

The interphase nucleus of the human eukaryotic cell, before DNA replication, contains 46 linear DNA molecules, each of the order of centimeters in length, in a spherical nucleus with a diameter of 3-10 microns. How does the cell avoid topological entanglements between the 92 linear DNA molecules following replication? A model of chromosome replication is introduced, based on the assumption of the existence of a physical linkage between diverging growing forks during eukaryotic chromosome replication. This basic model is shown to be sufficient for the avoidance of DNA duplex entanglements during DNA replication. The model also suggests structural characteristics of chromosomes at various points in the cell cycle and provides a possible partial mechanism for chromosome condensation at the end of replication.

Animals↗
PubMed1985

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.

Aspergillus nidulans↗
PubMed2003

Modelling study on the protective role of OH radical scavengers and DNA higher-order structures in induction of single- and double-strand break by gamma-radiation.

PURPOSE: To quantify the protective effects of (non-histonic) OH-radical scavengers and DNA higher-order structures in induction of single- (ssbs) and double-strand breaks (dsbs) by gamma-rays. MATERIALS AND METHODS: Spatial distributions of energy depositions by gamma-rays in liquid water were modelled with the track structure modules of the biophysical simulation code PARTRAC. Such distributions were superimposed on different DNA structure models (e.g. linear DNA, SV40 'minichromosomes' and compact chromatin), and direct energy depositions in the sugar-phosphate were considered as potential (direct) ssbs. The diffusion and interaction of the main chemical species produced in liquid water radiolysis were explicitly simulated, and reactions of *OH with the sugar-phosphate were considered as potential (indirect) ssbs. Two ssb on opposite DNA strands within 10 base pairs were considered as one dsb. Yields of ssb and dsb Gy(-1) Dalton(-1) in different DNA target structures were calculated as a function of the *OH mean lifetime, whose inverse value was taken as representative of the scavenging capacity of the DNA environment. RESULTS AND CONCLUSIONS: A further validation of the models implemented in the PARTRAC code has been provided, thus allowing a better understanding of the mechanisms underlying DNA damage. More specifically, the protection due to *OH scavengers was separately quantified with respect to that due to histones and chromatin folding, which could be 'switched off' in the simulations. As expected, for a given value of the environment scavenging capacity, linear DNA was more susceptible to strand breakage than SV40 minichromosomes, which in turn showed higher damage yields with respect to cellular DNA due to the larger accessibility offered to *OH. Furthermore, by increasing the scavenging capacity, the break yields decreased in all structures and tended to coincide with direct damage yields. Very good agreement was found with available experimental data. Comparisons with data on 'nucleoid' DNA (i.e. unfolded and histone-depleted DNA) also suggested that the experimental procedures used to obtain such structures might lower the environment scavenging capacity owing to the loss of cellular scavengers.

DNA↗
PubMed1996

The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats flanking the extremities of the linear chromosomal DNA.

Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering Asel fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a Dral map was obtained by positioning the Dral sites relative to the Asel map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the Asel and Dral fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The two terminal Asel fragments exhibited retarded pulsed-field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.

Chromosomes, Bacterial↗
PubMed1981

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus.

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.

Animals↗
PubMed1997

DNA binding properties of the hfq gene product of Escherichia coli.

We found that plasmids isolated by alkaline-lysis method occasionally showed abnormal mobility on agarose gel electrophoresis. This abnormality was not detected after phenol extraction of plasmids, indicating that it was caused by proteinous factor(s). A 15-kDa protein was found in the plasmid preparations on SDS-PAGE. The sequence of eighteen N-terminal amino acid residues of the 15-kDa protein was identical with the host factor I (HF-I) encoded by the hfq gene at 95 min on the Escherichia coli chromosome map, which is known to be required for bacteriophage Qbeta replication. The HF-I protein was purified and in vitro DNA binding experiment was carried out. HF-I bound to both supercoiled DNA and linear DNA and the binding of HF-I seemed to be sequence-nonspecific.

Amino Acid Sequence↗
PubMed1976

The effect of bleomycin on mitochondrial DNA.

Covalently closed circular mitochondrial DNA (mt-DNA) from mouse fibroblasts (L-cells) forms a separate band from nicked circular or linear DNA molecules in ethidium bromide-cesium chloride byoyant density gradient analysis. The conversion of covalently closed circular mt-DNA to nicked circular or linear DNA may serve as a sensitive method of detecting DNA strand breaks induced by bleomycin (BLM). At a very high concentration BLM induces the conversion of closed circular mt-DNA to nicked circular or linear DNA, however even after 24 h of exposure to BLM at a high concentration a considerable amount of closed circular mt-DNA molecules remains intact.

Animals↗
PubMed1986

Interaction between gene II protein and the DNA replication origin of bacteriophage f1.

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.

Bacteriophages↗
PubMed2006

Cloning the genes and DNA binding properties of High Mobility Group B1 (HMGB1) proteins from the human blood flukes Schistosoma mansoni and Schistosoma japonicum.

The parasitic helminth Schistosoma mansoni contains three HMGB proteins, HMGB1, HMGB2 and HMGB3, of primary amino acid sequences highly similar to vertebrate proteins. In this report we describe the characterization of the HMGB1 proteins and their genes from S. mansoni and Schistosoma japonicum. The deduced amino acid sequences of HMGB1 proteins from both schistosome species are identical, and comprise 176 residues. The proteins contain the two evolutionarily highly conserved HMG-box domains, A and B, exhibiting 60% similarity to mammalian HMGB1. Unlike the human HMGB1 which contains an unbroken run of 30 glutamic or aspartic residues, the SmHMGB1 or SjHMGB1 proteins possess unusually short acidic C-terminal tails (5 acidic residues interrupted by 2 serines). Southern hybridization and DNA sequencing revealed a single copy HMGB1 gene, composed of 3 exons and two introns, in S. mansoni. The exon/intron boundaries are identical to those of the human HMGB1 gene, with the exception that the second exon of the SmHMGB1 gene which is not split into two exons as in the human HMGB1 gene. RNA blot analysis revealed that the SmHMGB1 gene is constitutively expressed in similar levels both in male and female worms. The single-sized mRNA for SmHMGB1 is consistent with the size derived from the cDNA. Although DNA binding properties of SmHMGB1 (or SjHMGB1) protein seem to be similar to those previously reported with human HMGB1, i.e., preferential binding to supercoiled DNA over linear DNA, specific recognition of DNA four-way junctions, DNA-induced supercoiling in the presence of topoisomerase I, and DNA bending, we have observed two important differences relative to those observed with the human HMGB1: (i) the inability of the isolated SmHMGB1 domain A to bend DNA (as revealed by T4 ligase-mediated circularization assay), and (ii) higher DNA supercoiling and bending potential of the SmHMGB1 protein as compared to its human counterpart. The latter finding may indicate that the long acidic C-tail of human HMGB1 has much stronger repressive role on DNA bending or DNA supercoiling by topoisomerase I at physiological ionic strength than the short C-tail of the SmHMGB1 protein. Considering the important role of HMGB1 in DNA replication, transcription, recombination, and in particularly, the mediation of inflammation responses in mammalian cells, further studies on schistosome HMGB proteins may provide valuable information related to schistosomiasis, where inflammation plays a critical role in this disease.

Amino Acid Sequence↗
PubMed2001

In-vitro competition analysis of procyclin gene and variant surface glycoprotein gene expression site transcription in Trypanosoma brucei.

In Trypanosoma brucei, alpha-amanitin-resistant transcription characteristic of RNA polymerase I is initiated at ribosomal RNA gene (RRNA), procyclin gene (GPEET or EP1), and variant surface glycoprotein gene expression site (VSG ES) promoters. The three promoter types do not share obvious sequence homologies, but contain a proximal domain I and a distal domain II within 80 bp upstream of the transcription initiation site. RRNA, GPEET and EP1, but not the VSG ES promoter, require additional upstream sequences for full activity. In the present study, we competed in-vitro transcription of circular template DNA with linear DNA fragments to identify promoter domains responsible for binding and sequestering essential trans-acting transcription factors. For the GPEET promoter, we found that domain III, located between positions -141 and -92, was most important for the DNA fragment to exert a transcription competition effect, whereas domain I, the only element absolutely required for transcription, was not. Moreover, insertions between promoter domains II and III reduced both transcription from the GPEET promoter and competition with the GPEET promoter fragment, suggesting that these two domains cooperate in the formation of a stable DNA-protein complex. Taken together, these results indicate a promoter structure very similar to that of the Saccharomyces cerevisiae RRNA promoter. In contrast, VSG ES promoter analysis showed that domains I and II are both necessary and sufficient to compete transcription. Despite this structural difference, our analysis provide evidence that GPEET and VSG ES promoters interact with a common factor that is also important for RRNA promoter transcription.

Animals↗
PubMed2000

DNA repair patch-mediated double strand DNA break formation in human cells.

To investigate the mechanism of double strand DNA break formation in mammalian cells, an in vitro assay was established using closed circular DNA containing two uracils on opposite DNA strands (18 and 30 base pairs apart) and extracts prepared from human cells. In this assay, formation of double strand breaks was detected by the conversion of circular DNA to linear DNA. Approximately 4-fold more double strand DNA breaks were produced by extracts from cells deficient in DNA ligase I (46BR) relative to those produced by extracts from control cells (MRC5, derived from a clinically normal individual). In parallel with the amount of double strand DNA breaks, extracts from 46BR cells produced longer repair patches (up to 24 bases in length) than those from MRC5 cells (typically <5 bases long). When purified DNA ligase I was added to 46BR extracts to complement the DNA ligase deficiency, only a negligible difference was found between the amount of doublestrand DNA breaks or the repair patch size generated in the assay relative to MRC5 extracts. Together, our data demonstrate that double strand DNA breaks are produced through formation of DNA repair patches. We refer to this process of double strand break formation as the "DNA repair patch-mediated pathway."

Cell Line, Transformed↗
PubMed1999

Ca2+-dependent activity of human DNase I and its hyperactive variants.

We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double-stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300-fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild-type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+-dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild-type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26-fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate-enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.

Binding Sites↗
PubMed1993

Linear plasmids and chromosomes in bacteria.

Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram-positive bacteria, and Gram-negative bacteria. Two structural types of bacterial linear DNA have been characterized. Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram-positive filamentous Streptomyces have a covalently attached protein at each end. Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes. Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers. The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes.

Bacteria↗
PubMed1983

Transformation of intact yeast cells treated with alkali cations.

Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.

Cations, Monovalent↗
PubMed1972

Structural roles of polyoma virus proteins.

The superhelical, closed circular form of polyoma deoxyribonucleic acid (DNA) (Co 1) is bound in a 25S DNA-protein complex to the viral histone-like proteins after alkaline disruption of the virion. Nicked viral DNA or linear DNA are largely free of protein. Most of the viral protein disruption is in the form of capsomeres, sedimenting principally at 10S and 7S. Despite the relatively constant ratio of 10S to 7S material in many preparations, (1:5.5 to 1:6.0, respectively), the two classes of capsomeres are indistinguishable by electron microscopy and contain only P(2), P(3), and P(4) in molar ratios of approximately 5:1:1 or 6:1:1, respectively. Material with sedimentation rates of approximately 1 to 3S is enriched for P(5) and contains small amounts of P(2), P(3), and P(4). During the in vitro reassembly of DNA-free, shell-like particles from disrupted virus, proteins P(1), P(2), P(3), P(4), and P(7) are reincorporated efficiently, whereas P(5) and P(6) are not. The presence in empty reassembled particles of histone-like protein, expecially P(7), implies that at least this one of the minor protein components of the virion may participate in protein-protein interactions with other components of the capsid.

Autoradiography↗
PubMed1990

Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI.

Adenoviruses, Human↗
PubMed2003

[Detection of linear chromosomes and plasmids among 15 genera in the Actinomycetales].

Bacterial chromosomes and plasmids are commonly circular, however, linear chromosomes and plasmids were discovered among 5 genera of the Actinomycetales. Here, we use pulsed field gel electrophoresis to study the genomes of 19 species which belong to 15 genera in the Actinomycetales. All chromosomes of 19 species are linear DNA, and linear plasmids with different sizes and copy numbers are detected among 5 species. This work provide basis for investigating the possible novel functions of linear replicons beyond Streptomyces and also helps to develop Actinomycetales artificial linear chromosome.

Actinomycetales↗