L1 sequences in HeLa extrachromosomal circular DNA: evidence for circularization by homologous recombination.

Subcloned probes of the L1 family of repetitive elements were used to isolate L1-carrying clones from a plasmid library of HeLa cell extrachromosomal circular DNA. One clone was analyzed in detail by restriction mapping, cross-hybridization to L1 probes, and base sequence analysis. In addition to approximately the 3' half of a full-sized L1 element, this clone carried 390 base pairs of non-L1 sequence that is single copy in the HeLa genome. A HeLa genomic clone of this unique chromosomal region was isolated and the sequence organization of the circle clone was compared with the linear chromosomal region from which it was ultimately derived. We discuss possible mechanisms of circular DNA formation and propose homologous intrachromosomal recombination between 9-base-pair direct repeats to be most likely in this case.     

Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: evidence for a large specific deletion occurring at high frequency during circularization.

Rat hepatoma cells infected with mouse mammary tumor virus contain multiple forms of unintegrated viral DNA when grown in the presence of glucocorticoids. Using the DNA transfer procedure of Southern, we have prepared restriction endonuclease fragment maps of these forms of viral DNA. The maps indicate that: (i) the major species of viral DNA is a linear molecule of 5.9 X 10(6) Mr located in the cytoplasm; (ii) the nuclei contain covalently closed circular viral DNA of two distinct sizes (5.1 X 10(6) and 5.9 X 10(6) Mr) in addition to linear molecules (5.9 X 10(6) Mr); (iii) the linear molecule has specific termini; (iv) there is extensive homology between regions at or near termini of the linear molecule; (v) the predominant form of circular DNA lacks 1.2 kilobase pairs present in both the larger circular molecule and the linear molecule; and (vi) the sequences deleted from the majority of the circular DNA molecules are located at the ends of the linear DNA that are joined during circularization.     

Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA.

DNA can be chemically cleaved at the site of chloroacetaldehyde-modified residues by the chemicals used for Maxam-Gilbert sequencing reactions. Use of this technique facilitates fine structural analysis of unpaired DNA bases in DNA with non-B-DNA structure. This method was used to study the non-B-DNA structure adopted by the poly-(dG).poly(dC) sequence under torsional stress at various ionic conditions. In the presence of 2 mM Mg2+, the 5' half of the deoxycytosine tract is very reactive to chloroacetaldehyde, while the 3' half is virtually unreactive. In the poly(dG) tract, chloroacetaldehyde reaction is restricted to the center guanine residues. In the absence of Mg2+, however, it is the 5' half of the deoxyguanine tract that is reactive to chloroacetaldehyde, while the 3' half is unreactive. And chloroacetaldehyde reaction is restricted to the center cytosine residues in the poly(dC) stretch. These results strongly suggest that the poly(dG).poly(dC) sequence is folded into halves from the center of the sequence to form a tetra-stranded-like structure. Such a structure contains either a triplex consisting of poly(dG).poly(dG).poly(dC) strands in the presence of Mg2+ or a triplex consisting of poly(dC).poly(dG).poly(dC) strands in the absence of Mg2+. The fourth strand, not involved in triplex formation, is closely associated with the triplex and is positioned in such a way that DNA bases are exposed and freely accessible to the chloroacetaldehyde reaction.     

Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method.

The principle of a DNA cloning procedure that directionally generates genomic DNA fragments 50-2000 kilobases away from an initial probe is presented. The method depends on partial digestion of high molecular weight genomic DNA and subsequent ligation at very low concentration to generate covalent DNA circles. A library of the junction fragments from these circles can then be constructed. Biological or physical selection of the junction pieces can be achieved by incorporating a marker DNA fragment into the covalent circles. A 45-kilobase cosmid fragment has been successfully used to test the procedure. At appropriately low ligation concentrations (0.8 micrograms/ml), approximately equal to 90% of the ligated DNA is present as monomeric circles. Larger DNA fragments will require reducing the DNA concentration as the inverse square root of the DNA length. A suppressor tRNA gene has been tested as the selectable marker gene. Ligation of the digested circles into an amber-mutated lambda phage and propagation in a sup- host allows only the phage that contain junction fragments to produce plaques. Potential applications of this approach, such as mapping of complex genetic loci or moving from a linked gene toward a gene of interest, are presented and discussed.     

Reconstruction of a Streptomyces linear replicon from separately cloned DNA fragments: existence of a cryptic origin of circular replication within the linear plasmid.

We report here the reconstruction of a functional linear replicon, the 12-kilobase Streptomyces clavuligerus plasmid pSCL, from separate DNA fragments cloned in Escherichia coli on the pUC19 plasmid. Protein-free DNA molecules containing the full-length pSCL sequence, an internally inserted thiostrepton-resistance gene, and adventitious nucleotides external to the pSCL termini were introduced into Streptomyces lividans, where the synthesis and functional attachment of replication proteins occurred and pSCL was established as an extrachromosomal linear replicon. Transformation of S. lividans with uncut supercoilded pUC19/pSCL DNA from E. coli or with a circularized 8-kilobase internal fragment of pSCL yielded circular replicons, indicating the existence of a cryptic origin of circular replication within the linear plasmid. Insertion mutations at sites that prevented the replication of pSCL linear plasmids also interfered with its replication in the circular mode.     

"Hybrid" synapses formed by foreign innervation of parasympathetic neurons: a model for selectivity during competitive reinnervation.

Selectivity of synapse formation after nerve regeneration was tested in the parasympathetic cardiac ganglion of frogs (Rana pipiens). First, we tested the ability of somatic motor axons to establish synaptic connections with denervated ganglion cells by implanting the hypoglossus nerve into the vagotomized heart. After several weeks, stimulation of the implanted hypoglossus mediated a parasympathetic-like inhibition of the heart rate, and synaptic responses produced by hypoglossal stimulation were recorded intracellularly in ganglion cells. Light and electron microscopy indicated that implanted hypoglossal nerve terminals contacted parasympathetic ganglion cells only on their axons and not on the cell body (where most vagal synapses are found in control animals). Second, we tested whether regenerating vagal preganglionic axons would complete with foreign (hypoglossal) terminals for innervation of cardiac ganglion cells. We allowed the vagus nerve to regenerate in animals in which the implanted hypoglossus had established functional contacts with the cardiac ganglion. Vagal axons were able to reinnervate the heart and reestablish synaptic connections on the cell bodies of ganglion cells. Furthermore, functional transmission at the foriegn (hypoglossal) terminals disappeared concomitant with vagal reinnervation.     

Molecular structure of nicked DNA: a substrate for DNA repair enzymes.

The molecular structure of a nicked dodecamer DNA double helix, made of a ternary system containing d(CGCGAAAACGCG) + d(CGCGTT) + d(TTCGCG) oligonucleotides, has been determined by x-ray diffraction analysis at 3 A resolution. The molecule adopts a B-DNA conformation, not unlike those found in intact dodecamer DNA molecules crystallized in a somewhat different crystal lattice, despite a gap due to the absence of a phosphate group in the molecule. The helix has a distinct narrow minor groove near the center of the molecule at the AAAA region. This suggests that the internal stabilizing forces due to base stacking and hydrogen-bonding interactions are sufficient to overcome the loss of connectivity associated with the disruption of the covalent backbone of DNA.     

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

When defined-sequence DNA from the lacl region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues. In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used. Chromophore-induced release of free cytosine base from [3H]cytosine-labeled DNA was 2- to 3-fold greater with glutathione than with the other thiols. These results suggest that the alkali-dependent strand break is some form of apyrimidinic site. These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA. These sites were also less sensitive to E. coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E. coli endonuclease III by a factor of at least 10. These and other results suggest that these sites are chemically different from normal apurinic/apyrimidine sites. When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacl gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A X T transitions at the same sites. All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites. The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G X C to A X T transitions in the mutational spectrum of neocarzinostatin.     

Promotion of parallel DNA quadruplexes by a yeast telomere binding protein: a circular dichroism study.

Repressor-activator protein 1 (RAP1) has an essential role in the maintenance of yeast telomeres. Yeast telomeric DNA consists of simple repeated G-rich sequences that are bound by RAP1. We have found that RAP1, in addition to its known binding activity for double-stranded DNA, interacts with the G-rich strand containing guanine base (G)-tetrads. We show here using circular dichroism spectroscopy that RAP1 promotes the formation of one particular type of DNA quadruplex, parallel G4-DNA. Furthermore, RAP1 is able to bind to both preformed parallel and antiparallel DNA quadruplexes. These results have implications for the possible use of DNA quadruplexes in telomere-telomere association in vivo.     

Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: the target of the drugs is DNA.

Norfloxacin is a nalidixic acid analogue and one of the most potent DNA gyrase inhibitors. To study the mechanism of this important class of inhibitors, the binding of [3H]norfloxacin to gyrase and substrate DNA was measured. We found that, contrary to prior belief, norfloxacin does not bind to gyrase but instead binds to DNA. This was demonstrated by both equilibrium dialysis and membrane filtration techniques. Binding to ColE1 and pBR322 plasmids showed a primary process that is saturated at a norfloxacin concentration about equal to its supercoiling Ki (1.8 X 10(-6) M) and is followed by weaker secondary binding. The apparent Kd values are 1 X 10(-6) M for both plasmids. The molar binding ratio at this initial saturation point is extremely low: only 4 X 10(-4) norfloxacin per nucleotide for both plasmids. The binding of norfloxacin to DNA plasmids is nonintercalative, as shown by the fact that the drug binds preferentially to single-stranded DNA rather than to double-stranded DNA. The binding is reduced at high salt concentration, has a pH optimum between 4.5 and 6.5, and does not require divalent ions. The binding affinities of other nalidixic acid analogues were estimated by an indirect competition method. The calculated apparent Kd values of these analogues correlate well with their Ki values, providing strong evidence that the binding affinity of the drug to DNA determines biological potency.     

Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications.

Inordinate expansion and hypermethylation of the fragile X DNA triplet repeat, (GGC)n.(GCC)n, are correlated with the ability of the individual G- and C-rich single strands to form hairpin structures. Two-dimensional NMR and gel electrophoresis studies show that both the G- and C-rich single strands form hairpins under physiological conditions. This propensity of hairpin formation is more pronounced for the C-rich strand than for the G-rich strand. This observation suggests that the C-rich strand is more likely to form hairpin or "slippage" structure and show asymmetric strand expansion during replication. NMR data also show that the hairpins formed by the C-rich strands fold in such a way that the cytosine at the CpG step of the stem is C.C paired. The presence of a C.C mismatch at the CpG site generates local flexibility, thereby providing analogs of the transition to the methyltransferase. In other words, the hairpins of the C-rich strand act as better substrates for the human methyltransferase than the Watson-Crick duplex or the G-rich strand. Therefore, hairpin formation could account for the specific methylation of the CpG island in the fragile X repeat that occurs during inactivation of the FMR1 gene during the onset of the disease.     

Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.

A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.     

In vitro mutagenesis of a circular DNA molecule by using synthetic restriction sites.

A method for mutagenizing circular DNA molecules has been developed that uses synthetic oligodeoxynucleotide restriction sites as mutagens. A single synthetic restriction site is introduced at random by cleaving circular DNA with a nonspecific double-strand endonuclease. The restriction site is then ligated to the ends and the molecule is subsequently recircularized. These small additions to the genome are mapped by digestion with the appropriate restriction enzyme. Rearrangements such as duplications and deletions can be engineered at will by using the added restriction sites. This technique has been used to produce a fine-structure map of RSF1050, a ColE1 derivative, 60% of which is a transposable DNA sequence encoding the TEM beta-lactamase (Tn3). A subset of the mutations, mapping within a narrow region of Tn3, result in an increased frequency of Tn3 transposition; mutations in other regions abolish transposition entirely.     

Rapid kinetics of mismatch repair of heteroduplex DNA that is formed during recombination in yeast.

Homothallic switching of yeast mating type (MAT) genes is a highly efficient gene conversion process initiated by a double-strand break. The use of a galactose-inducible HO endonuclease gene has made it possible to analyze the synchronous progression of molecular intermediates during recombination. When MATa switches to MAT alpha, a 3' single-stranded end of HO-cleaved MAT DNA invades the homologous donor, HML alpha, and initiates copying of new DNA sequences. These early steps of recombination can be detected by PCR amplification. When recombination is initiated in a strain carrying the MATa-stk T-->A base pair substitution mutation located 8 bp to the right of the HO endonuclease cleavage site, the stk mutation is frequently included in heteroduplex DNA formed between MAT and HML and undergoes mismatch correction. We have followed the kinetics of mismatch repair of the stk mutation by determining the DNA sequence of the PCR-amplified early intermediates of recombination. Mismatch correction of heteroduplex DNA is quite rapid (t1/2 = 6-10 min) compared to the 60 min required to complete repair of the double-strand break. Mismatch repair occurs soon after the 3'-ended MAT-stk strand invades HML and forms heteroduplex DNA. Moreover, nearly all the correction events are restorations, in which the invading MAT-stk strand is corrected to the genotype of the resident HML donor. This rapid restoration ensures that the net result will be a gene conversion at the MAT locus. Rapid and preferential mismatch repair of heteroduplex DNA has important implications in understanding meiotic recombination.     

Replication of linear mitochondrial DNA from Paramecium: sequence and structure of the initiation-end crosslink.

Replication of the 14-micrometer linear Paramecium mitochondrial DNA is initiated by a crosslinking of the duplex strands at the initiation end of the molecule. As a consequence of the crosslink, a head-to-head dimer molecule, or palindrome, is a replicative intermediate. The central region of the dimer molecules of two species was cloned and sequenced, In the distal regions, the sequence is palindromic as expected; however, in the central region, there is a nonpalindromic sequence that is rich in A + T and contains direct tandem repeats. It is proposed that the nonpalindromic sequence constitutes the crosslink. Implications for the replication scheme are discussed. The model is unlike any other for a linear DNA.     

Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure.

This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.     

The structure of DNA in a nucleosome.

We describe the application of the hydroxyl radical footprinting technique to examine the histone-DNA interactions of a nucleosome that includes part of the 5S ribosomal RNA gene of Xenopus borealis. We establish that two distinct regions of DNA with different helical periodicities exist within the nucleosome and demonstrate a change in the helical periodicity of this DNA upon nucleosome formation. In particular, we find that on average the helical periodicity of DNA in this nucleosome is 10.18 +/- 0.05 base pairs per turn. The same DNA, when bound to a calcium phosphate surface, has a periodicity of 10.49 +/- 0.05 base pairs per turn, similar to that of random sequence DNA. Modulations in minor groove width within the naked DNA detected by the hydroxyl radical are maintained and exaggerated in nucleosomal DNA. These features correlate with regions in the DNA previously suggested to be important for nucleosome positioning.