Prep-A-Gene: a superior matrix for the purification of DNA and DNA fragments

A new chromatographic matrix, Prep-A-Gene, is described for the isolation and purification of high quality DNA suitable for restriction analysis, ligation, transformation and sequencing protocols. This matrix selectively binds DNA greater than approximately 200 base pairs in length, while RNA, proteins, cellular components, agarose and other contaminants are washed free in minutes. This eliminates the need for time-consuming and laborious RNase treatments, gel extractions and phenol extractions. The DNA that is desorbed from the matrix is available immediately as a substrate for subsequent protocols. DNA purified in this manner exhibits no detectable shearing, even with more fragile chromosomal DNA.     

DNA replication as a target of the DNA damage checkpoint

Faithful inheritance of the genome from mother to daughter cell requires that it is replicated accurately, in its entirety, exactly once. DNA replication not only has to have high fidelity, but also has to cope with exogenous and endogenous agents that damage DNA during the life cycle of a cell. The DNA damage checkpoint, which monitors and responds to defects in the genome, is critical for the completion of replication. The focus of this review is how DNA replication is regulated by the checkpoint response in the presence of DNA damage and fork stalling agents.     

Cooperative DNA Binding and Protein/DNA Fiber Formation Increases the Activity of the Dnmt3a DNA Methyltransferase

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.     

DNA synthesis at a fork in the presence of DNA helicases

In a mixture of Escherichia coli DNA polymerase III holoenzyme, single-strand-binding protein, artificially forked lambda bacteriophage DNA with primer annealed to the leading side of the fork, dNTPs and ATP, DNA synthesis is enhanced by helicase II, less so by helicases, I, III or rep protein of E. coli or T4 phage helicase. The effect of helicase II depends on ATP, it is enhanced by helicase III, and it is not observed using DNA polymerase I or T4 DNA polymerase. In the absence of dNTPs helicase II is less active than helicase I or T4 helicase in unwinding the forked DNA. We believe that helicase II both shifts the forks and stimulates DNA polymerase III. The results support the conclusion derived from previous studies that helicase II is part of the DNA-synthesizing system of E. coli.     

DNA–DNA kissing complexes as a new tool for the assembly of DNA nanostructures

Kissing-loop annealing of nucleic acids occurs in nature in several viruses and in prokaryotic replication, among other circumstances. Nucleobases of two nucleic acid strands (loops) interact with each other, although the two strands cannot wrap around each other completely because of the adjacent double-stranded regions (stems). In this study, we exploited DNA kissing-loop interaction for nanotechnological application. We functionalized the vertices of DNA tetrahedrons with DNA stem-loop sequences. The complementary loop sequence design allowed the hybridization of different tetrahedrons via kissing-loop interaction, which might be further exploited for nanotechnology applications like cargo transport and logical elements. Importantly, we were able to manipulate the stability of those kissing-loop complexes based on the choice and concentration of cations, the temperature and the number of complementary loops per tetrahedron either at the same or at different vertices. Moreover, variations in loop sequences allowed the characterization of necessary sequences within the loop as well as additional stability control of the kissing complexes. Therefore, the properties of the presented nanostructures make them an important tool for DNA nanotechnology.     

Counting DNA: estimating the complexity of a test tube of DNA

We consider the problem of estimation of the 'complexity' of a test tube of DNA. The complexity of a test tube is the number of different kinds of strands of DNA in the test tube. It is quite easy to estimate the number of total strands in a test tube, especially if the strands are all the same length. Estimation of the complexity is much less clear. We propose a simple kind of DNA computation that can estimate the complexity.     

Identification of a DNA segment containing the human DNA polymerase alpha gene

We isolated a temperature-sensitive mutant from mouse FM3A cells, designated as tsFT20, the DNA polymerase alpha activity of which is heat-labile. A hybrid clone (M6-39 cells) between human cells and tsFT20 cells contained one or two human chromosomes. M6-39 cells (primary hybrid) were exposed to gamma-ray and re-fused with tsFT20, after which we isolated two temperature-resistant secondary hybrids, both of which retained an identical minute portion of the human chromosome, 400-500 kilobase pairs (kbp). Immunological studies demonstrated that this secondary hybrid expressed human DNA polymerase alpha. Thus, the human DNA polymerase alpha gene was located within a DNA region of 400-500 kbp.     

Biological consequences of a reduction in the non-target DNA scanning capacity of a DNA repair enzyme

Numerous DNA-interactive proteins have been shown to locate specific sequences within large domains of non-target DNA in vitro and in vivo by a one-dimensional diffusion mechanism; however, the biological significance of this process has not been evaluated. We have examined the biological consequences of sliding for the pyrimidine dimer-specific DNA repair enzyme T4 endonuclease V, an enzyme which scans non-target DNA both in vitro and in vivo. An endonuclease V mutant was constructed whose only altered biochemical characteristic, measured in vitro, was a loss in its ability to slide on non-target DNA. In contrast to the native enzyme, when the mutated endonuclease V was expressed in DNA repair-deficient Escherichia coli, no enhanced ultraviolet survival was conferred. These results suggest that the mechanisms which DNA-interactive proteins employ to enhance the probability of locating their target sequences are of significant biological importance.     

Modeling the mechanics of a DNA oligomer

DNA stretching and strand separation have been studied by molecular mechanics using an oligomer which has been the subject of nanomanipulation experiments (Noy et al., Chem. Biol. 4, 519, 1997). Adiabatic mapping of conformational energy carried out as a function of stretching leads to force/extension curves in good correlation with the experimental results. Other types of deformation are also modeled and compared with the experimental results obtained on polymeric DNA. The results highlight overall similarities, but point to thermodynamic differences and also to local base sequence effects which can be expected to play an important role at the level of biologically induced structural deformations.     

At a crossroads: human DNA tumor viruses and the host DNA damage response

Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection.     

DNA sequence of a Salmonella-specific DNA fragment and the use of oligonucleotide probes for Salmonella detection

Summary Hybridization specificity of a 1.8-kb HindIII DNA fragment isolated from Salmonella typhimurium by a molecular cloning technique was confirmed by colony hybridization with 327 Salmonella isolates of various serotypes and 56 non-Salmonella isolates including Enterobacteriaceae closely related to Salmonella, such as Escherichia coli, Klebsiella, Citrobacter and Shigella. It was found that this 1.8-kb DNA fragment was highly specific for all the Salmonella isolates tested. The DNA sequence of this 1.8-kb fragment was then determined by the dideoxynucleotide chain termination method. According to this DNA sequence, six oligonucleotide fragments ranging from 17- to 26-mer were then chemically synthesized and tested for their hybridization specificities. Results show that three of the six oligonucleotide fragments are highly specific for all 327 Salmonella strains tested and can be used as probes for the specific detection of Salmonella in foods or other samples. Offprint requests to: H.-Y. Tsen     

On the shape of a DNA molecule

Due to the lack of direct X-ray evidence for base pairing being the only mechanism for the formation of double helix in a DNA crystal, an alternative explanation is suggested so that the observed DNA loop becomes essential. This work was sponsored at Brown University under a grant from Speidel Corporation.     

Size fractionation of Trypanosoma brucei DNA: localization of the 177-bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction

We have size-fractionated intact DNA from Trypanosoma brucei into a major large DNA fraction (greater than 350S) and minor middle-sized (60-250S) and small (less than 60S) DNA fractions. Large DNA contains the rRNA genes, the basic copy genes for several variant surface glycoproteins (VSGs), including one which lies near a telomer, and the expression-linked copies of the two VSG genes. The middle-sized DNA contains at least one VSG gene, but the hybridization of this fraction with probes for the conserved repetitive sequences that mark the edges of the transposed segments of VSG genes, suggests that it may contain many VSG genes. The 177-bp repeat satellite DNA is also exclusively found in this fraction.     

Effect of imino group of a linker arm at the C5 position of a pyrimidine nucleoside on the thermal stabilities of DNA/DNA and DNA/RNA duplexes

The modified ODN's bearing C5-substituted 2'-deoxyuridine derivative were synthesized by a post-synthetic modification with an unsymmetrical triamine. The effect of the C5-substituent on the duplex formation with complementary DNA or RNA differed with the position of an imino group in the linker-arms.     

Effect of Imino Group of a Linker Arm at the C5 Position of a Pyrimidine Nucleoside on the Thermal Stabilities of DNA/DNA and DNA/RNA Duplexes

The modified ODN's bearing C5‐substituted 2′‐deoxyuridine derivative were synthesized by a post‐synthetic modification with an unsymmetrical triamine. The effect of the C5‐substituent on the duplex formation with complementary DNA or RNA differed with the position of an imino group in the linker‐arms.