Cohesive Termini Research Articles

Complete genome sequence of the novel phage vB_EcoS_PHB17, which infects Shiga-toxin-producing Escherichia coli.

The complete genome of the novel phage vB_EcoS_PHB17, which infects Shiga-toxin-producing Escherichia coli, was sequenced, revealing a linear double-stranded DNA genome of 48,939bp with 46% GC content and protruding 150-bp 5' cohesive termini. The genome contained 85 open reading frames, 28 of which were annotated with known functions. No tRNA-encoding genes were detected. Phylogenetic analysis suggested that phage PHB17 is a novel phage of family Siphoviridae.

MCT cloning: a seamless cloning strategy for inserting DNA fragments

This study presents a seamless, efficient and restriction/ligation-independent cloning strategy for inserting large fragments: MCT cloning (based on the generation of Manmade Cohesive Termini). This method requires only four steps: (i) the first parallel PCR for the amplification of complete and truncated insert fragments; (ii) the second parallel PCR for the exponential amplification of linear plasmids with the same sequences and different break sites mediated by the first products and the corresponding reverse primers; (iii) the formation of manmade cohesive termini after DpnI digestion, mixing, denaturation and annealing; and (iv) transformation. By employing this strategy, large fragments (1–4 kbp) can be easily inserted into the pGADT7 vector (∼ 8 kbp).

Properties of an NAD +-dependent DNA ligase from the hyperthermophile Thermotoga maritima and its application in PCR amplification of long DNA fragments

The thermostable DNA ligase of Thermotoga maritima ( Tma DNA ligase) was expressed in Escherichia coli, and purified by heat treatment followed by metal affinity chromatography. Purified Tma DNA ligase exhibited activity on DNA fragments with cohesive termini, and no activity was detected on blunt-end DNA. The ligase reaction required NAD +, and a divalent cation including Mg 2+, Mn 2+or Ca 2+. The highest activity of Tma DNA ligase occurred at 60 °C and pH 8.0 when Hind III digested plasmid was used as substrate. The purified enzyme had a half-life of over 30 min at 95 °C, and retained over 80% of its activity after holding a pH ranging from 7.2 to 8.8 for 1 h at 80 °C. When the enzyme was employed in PCR cycles, Tma DNA ligase promoted the amplification of long DNA fragments from the genomic DNA of T. maritima.

Haloviruses HF1 and HF2: evidence for a recent and large recombination event.

Haloviruses HF1 and HF2 were isolated from the same saltern pond and are adapted to hypersaline conditions, where they infect a broad range of haloarchaeal species. The HF2 genome has previously been reported. The complete sequence of the HF1 genome has now been determined, mainly by PCR and primer walking. It was 75,898 bp in length and was 94.4% identical to the HF2 genome but about 1.8 kb shorter. A total of 117 open reading frames and five tRNA-like genes were predicted, and their database matches and characteristics were similar to those found in HF2. A comparison of the predicted restriction digest patterns based on nucleotide sequence with the observed restriction digest patterns of viral DNA showed that, unlike the case for HF2, some packaged HF1 DNA had cohesive termini. Except for a single base change, HF1 and HF2 were identical in sequence over the first 48 kb, a region that includes the early and middle genes. The remaining 28 kb of HF1 showed many differences from HF2, and the similarity of the two genomes over this late gene region was 87%. The abrupt shift in sequence similarity around 48 kb suggests a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb. This example indicates there is a high level of recombination among viruses that live in this extreme environment.

A generalized transducing thiophage (TPC-1) of a facultative sulfur chemolithotrophic bacterium, Bosea thiooxidans CT5, of α- Proteobacteria, isolated from Indian soil

We have isolated and characterized a double-stranded DNA bacteriophage (TPC-1) of Bosea thiooxidans, a facultative sulfur chemolithotrophic bacterium. The name ‘thiophage’ is introduced for phage(s) infecting sulfur chemolithotrophic bacteria. Electron micrographs showed the phage particle with an icosahedral head and a very short wedge-like tail. TPC-1 is classified as the C1 morphotype of the Podoviridae family. Restriction map and terminal ends detection by end fill labeling of the TPC-1 genomic DNA showed that the genome is linear with 5′ protruding cohesive termini. Contour length mapping of the DNA genome also revealed it to be a linear fragment with size (∼44 kb) corresponding with the size estimated from restriction fragment analyses and proved the non-redundant nature of the linear genome topology. In colorless sulfur chemolithotrophic microorganisms, TPC-1 is the first report of a generalized transducing thiophage.

Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.

A novel DNA-binding protein associated with DNA polymerase-alpha in pea stimulates polymerase activity on infrequently primed templates.

A 42 kDa DNA-binding protein is associated with DNA polymerase-alpha-primase in pea (Pisum sativum). In a previous publication it was shown that the protein has strong preference for ds-ss junctions in DNA, including the cohesive termini generated by restriction endonucleases. In this paper it is shown that when the DNA-binding protein is added back to polymerase-primase, the protein stimulates the activity of the polymerase. The stimulation is particularly marked when M13 DNA, primed with a single sequencing primer or primed with oligoribonucleotides by the polymerase's associated primase activity, is used as a template. The stimulation of polymerase activity is not caused by an increase in processivity. These data lead to the suggestion that the 42 kDa DNA-binding protein is a primer-recognition protein.

Mouse testicular extracts process DNA double-strand breaks efficiently by DNA end-to-end joining

DNA double-strand break (DSB) processing was studied in mouse testicular extracts using a defined DSB created by cleaving supercoiled pUC12 DNA at a unique site as the substrate, and analysing the processed DNA by gel electrophoresis. Our results demonstrated that enzymatic activity in the extracts promoted multimerization of DNA and suppressed its circularization. This was distinctly different from T4 DNA ligase activity in the control and therefore the process must be more complex than simple ligation. Efficiency of this end-to-end joining was ATP and Mg2+-dependent and was much higher with cohesive (especially with 5′) than with blunt ends. On recleaving, the joining was predominantly faithful, especially for cohesive ends; but a detectable fraction of DNA had undergone end-processed joining causing junctional deletions, mostly with blunt ends. Redigestion of end-joined products from time course experiments established that the end-deleted joining occurred concurrent to the faithful joining. Junctional segments were cloned and their restriction analysis confirmed the presence of large deletions from both the sides. These results suggested the association of an end-processing activity (exonuclease/helicase+flap endonuclease) along with the end-joining ligase(s). Suppression of end-edited joining on lowering the reaction temperature to 17° or 14°C, despite efficient faithful joining, indicated that this enzymatic activity is retarded at low temperature. Though the efficiency and fidelity of joining were termini-dependent, the orientation of joining was random. Lack of preference for homologous ends (H:H or T:T), as well as efficient joining of heterologous DNA (pUC12/pBR322) having two different blunt termini, showed that the end joining could occur independent of extensive/terminal homology. Retention of radioactivity on end joining of (α-32P)dCTP end-filled cohesive termini, and lack of their junctional cleavability, apparently due to restriction site duplication, suggested direct double strand ligation. Thus it is demonstrated that mouse male germ cells possess an efficient DNA end-joining activity, involving either a major pathway of precise joining, or a minor end-deleted joining, and it seems to be achieved by a multienzymatic complex as suggested also for somatic cells by others. These results show that mammalian male germ cells that are proficient in homologous recombination utilize nonhomologous end-joining (NHEJ) mechanism for DSB processing and therefore NHEJ is a conserved, universal pathway for the vital function of DSB repair.

Use of T4 DNA polymerase to create cohesive termini in PCR products for subcloning and site-directed mutagenesis.

Use of T4 DNA polymerase to create cohesive termini in PCR products for subcloning and site-directed mutagenesis.

DNA Bending Is Essential for the Site-specific Recognition of DNA Response Elements by the DNA Binding Domain of the Tumor Suppressor Protein p53

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.

Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways.

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.

The complete nucleotide sequence of bacteriophage HP1 DNA.

The complete nucleotide sequence of the temperate phage HP1 of Haemophilus influenzae was determined. The phage contains a linear, double-stranded genome of 32 355 nt with cohesive termini. Statistical methods were used to identify 41 probable protein coding segments organized into five plausible transcriptional units. Regions encoding proteins involved in recombination, replication, transcriptional control, host cell lysis and phage production were identified. The sizes of proteins in the mature HP1 particle were determined to assist in identifying genes for structural proteins. Similarities between HP1 coding sequences and those in databases, as well as similar gene organizations and control mechanisms, suggest that HP1 is a member of the P2-like phage family, with strong similarities to coliphages P2 and 186 and some similarity to the retronphage Ec67.

Condensation and cohesion of λ DNA in cell extracts and other media: Implications for the structure and function of DNA in prokaryotes

DNA added to concentrated extracts of Escherichia coli undergoes a reversible transition to a readily-sedimentable (‘condensed’) form. The transition occurs over a relatively small increment in extract concentration. The extract appears to play two roles in this transition, supplying both DNA-binding protein(s) and a crowded environment that increases protein binding and favors compact DNA conformations. The two roles of the extract are suggested by properties of fractions prepared by absorption of extracts with DNA-cellulose. The DNA-binding fraction and the DNA-nonbinding fractions from these columns are separately poorer condensing agents than the original extract, but when rejoined are similar to the original extract in the amount required for condensation. The dual role for the extract is supported by model studies of condensation with combinations of purified DNA-binding materials (protein HU or spermidine) and concentrated solutions of crowding agents (albumin or polyethylene glycol 8000); in each case, crowding agents and DNA-binding materials jointly reduce the amounts of each other required for condensation. The condensation reaction as studied in extracts or in the purified systems may be a useful approach to the forces which stabilize the compact form of DNA within the bacterial nucleoid. The effect of condensation on the reactivity of the DNA was measured by changes in the rate of cohesion between duplex DNA molecules bearing the complementary single-strand termini of λ DNA. Condensation caused large increases in the rates of cohesion of both λ DNA and of restriction fragments of λ DNA bearing the cohesive termini. Cohesion products of λ DNA made in vitro are a mixture of linear and circular aggregates, whereas those made in vivo are cyclic monomers. We suggest a simple mechanism based upon condensation at the site of viral injection which may explain this discrepancy.

Nonenzymatic plasmid ligation mediated by minor groove-binding molecules.

DNA ligation is the weak link in the chain of gene cloning. We have developed a straightforward nonenzymatic alternative to this reaction that employs easily available commercial reagents. The method uses the affinity of distamycin for the minor groove to join DNA ends together. Phosphodiester bonds are formed after cyanoimidazole-promoted phosphate activation in the presence of manganese(II) cations. When transfected into eukaryotic cells, the chemically ligated plasmid is transcribed even more efficiently than after enzymatic ligation. At present this technique compares favorably with T4 ligation for AT-rich cohesive termini, but in principle it could be extended to any restriction site.

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2.

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3' extended DNAs, with the following sequence: 5'-GTTAGGCTT-3' 3'-CAATCCGAA-5'. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3' extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5' extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.

Enhanced chromosomal response of ataxia-telangiectasia cells to specific types of DNA double-strand breaks

The chromosomal response of two ataxia-telangiectasia (A-T) lymphoblastoid cell lines (A-T-PA and A-T-KM) to restriction endonucleases (RE) is compared with that of a normal (N-SW) lymphoblastoid cell line. The RE used were PvuII (generating DNA double-strand breaks with blunt termini), BamHI (cohesive termini with 4 base, 5' overhangs) and PstI (cohesive termini with 4 base 3' overhangs). Chromatid aberrations were analysed in cells 5 h after treatment. Cells were porated using streptolysin O to allow entry of RE. Both A-T lines showed an enhanced frequency of chromatid breaks in G2 phase compared with normal cells in response to RE. The enhanced response of A-T cells was most marked in the case of PvuII treatment when the enhancement ratios were 2.5 and 4.2 for A-T-PA and A-T-KM respectively. However, the frequency of DNA double-strand breaks (dsb), measured by neutral filter elution, were considerably lower in A-T-PA cells than N-SW, due to a lower efficiency of poration. When A-T-PA cells were treated with streptolysin O at a higher concentration (0.3 Units/ml), a condition that apparently led to a similar level of poration in A-T-PA as in N-SW cells treated with 0.06 Units/ml as judged by the similar number of dsb induced in the two lines for a given PvuII concentration, the enhancement ratio for A-T-PA cells treated with PvuII increased from 2.5 to 5.8. BamHI and PstI were found to be less clastogenic in all three cell lines as found previously for Chinese hamster cells, although part of this effect may be due to a lower activity, particularly in the case of PstI. However, even at a 4-6-fold higher concentration, BamHI was still less clastogenic than PvuII. It is concluded that dsb with blunt termini are more clastogenic than those with cohesive termini. The results suggest that the chromosomal sensitivity of A-T cells may result from a defect causing a higher rate of conversion of dsb into chromatid aberrations.

Enhanced Chromosomal Response of Ataxia-telangiectasia Cells to Specific Types of DNA Double-strand Breaks

The chromosomal response of two ataxia-telangiectasia (A-T) lymphoblastoid cell lines (A-T-PA and A-T-KM) to restriction endonucleases (RE) is compared with that of a normal (N-SW) lymphoblastoid cell line. The RE used were PvuII (generating DNA double-strand breaks with blunt termini), BamHI (cohesive termini with 4 base, 5' overhangs) and PstI (cohesive termini with 4 base 3' overhangs). Chromatid aberrations were analysed in cells 5 h after treatment. Cells were porated using streptolysin O to allow entry of RE. Both A-T lines showed an enhanced frequency of chromatid breaks in G2 phase compared with normal cells in response to RE. The enhanced response of A-T cells was most marked in the case of PvuII treatment when the enhancement ratios were 2.5 and 4.2 for A-T-PA and A-T-KM respectively. However, the frequency of DNA double-strand breaks (dsb), measured by neutral filter elution, were considerably lower in A-T-PA cells than N-SW, due to a lower efficiency of poration. When A-T-PA cells were treated with streptolysin O at a higher concentration (0.3 Units/ml), a condition that apparently led to a similar level of poration in A-T-PA as in N-SW cells treated with 0.06 Units/ml as judged by the similar number of dsb induced in the two lines for a given PvuII concentration, the enhancement ratio for A-T-PA cells treated with PvuII increased from 2.5 to 5.8. BamHI and PstI were found to be less clastogenic in all three cell lines as found previously for Chinese hamster cells, although part of this effect may be due to a lower activity, particularly in the case of PstI. However, even at a 4-6-fold higher concentration, BamHI was still less clastogenic than PvuII. It is concluded that dsb with blunt termini are more clastogenic than those with cohesive termini. The results suggest that the chromosomal sensitivity of A-T cells may result from a defect causing a higher rate of conversion of dsb into chromatid aberrations.

A new restriction endonuclease, TspEI, from the genus Thermus that generates cohesive termini compatible with those of EcoRI.

The detection, isolation and properties of the restriction endonuclease TspEI are described. The canonical recognition sequence (AATT) is the same as the 4-bp core of the 6-bp sequence (GAATTC) of EcoRI. Hydrolysis occurs 5' to the palindromic tetramer so that TspEI produces the same cohesive termini as EcoRI. TspEI therefore has an obvious application in producing partial digests of DNA for ligation to EcoRI-digested cloning vectors.

Cohesive single-stranded ends of Streptomyces temperate bacteriophage R4

The cohesive single-stranded termini of temperate Streptomyces phage R4 were found to be complementary 11 base single-stranded 3′-extended DNAs with the sequence: 5′-CGCCGTGTCTT-3′ 3′-GCGGCACAGAA-5′

Salmonella phage PSP3, another member of the P2-like phage group

Freshly isolated DNA of phage PSP3, whose morphology closely resembles that of phage P2, contained both circular and linear molecules about 31 kb in length. Linear PSP3 DNA molecules possess single-stranded cohesive termini ( cos). Sequencing of the fragment anticipated to contain cos revealed a 19-base sequence identical to cos of phage 186. Of the 107 by to the right of cos, 94 were identical in 186 DNA (88% similarity), and of the 370 by to the left, 229 were identical (62% similarity). Cos flanking sequences in both P2 and P4 were also highly conserved in PSP3. A number of restriction sites were at similar locations on the two phage DNAs. The parasitic phage P4 propagated on PSP3 lysogens. PSP3 integrates into the Escherichia coli chromosome at 27 min.