Telomere shortening is proportional to the size of the G-rich telomeric 3'-overhang.

Most normal diploid human cells do not express telomerase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the length of the single-stranded G-rich telomeric 3'-overhang is proportional to the rate of shortening in four human cell types that exhibit different rates of telomere shortening in culture. These results provide direct evidence that the size of the G-rich overhang is not fixed but subject to regulation. The potential ability to manipulate this rate has profound implications both for slowing the rate of replicative aging in normal cells and for accelerating the rate of telomere loss in cancer cells in combination with anti-telomerase therapies.

DNA-sequence asymmetry directs the alignment of recombination sites in the FLP synaptic complex.

The FLP recombinase promotes site-specific recombination in the 2 micrometer circle of Saccharomyces cerevisiae. FLP recognizes a 48 bp target site (FLP recombination target, or FRT) consisting of three 13 bp protein binding sites, or symmetry elements, flanking an 8 bp spacer region. Efficient recombination also occurs with DNA substrates that have minimal FRT sites, consisting only of the spacer and two surrounding 13 bp symmetry elements arranged in inverse orientation; thus, the wild-type spacer sequence is the main asymmetric feature of the minimal recombination site. FLP carries out recombination with many minimal target sites bearing symmetric or asymmetric mutant spacer sequences; however, the overall directionality of recombination defined in terms of inversion or excision of a DNA domain is determined by spacer-sequence asymmetry. In order to evaluate the potential influence of spacer-sequence asymmetry on structures formed during early steps in recombination, we used electron microscopy to investigate the structure of the FLP synaptic complex, which is the intermediate protein-DNA complex involved in site pairing and strand exchange. Using linear substrate DNAs that have minimal FRTs with wild-type spacer sequences, we find that 85 to 90% of the FLP synaptic complexes examined contain the two FRTs aligned in parallel. This strong preference for parallel site alignment stands in contrast with prevailing models for lambda integrase-class recombination systems, which postulate antiparallel site alignment, and results from biophysical studies on synthetic, immobile four-way DNA junctions. Our results show that the strong preference for parallel alignment can be attributed to conformational preferences of Holliday junctions present in the synaptosome.

Normal human chromosomes have long G-rich telomeric overhangs at one end.

Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200 +/- 75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3' overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.

Supercoiling-dependent flexibility of adenosine-tract-containing DNA detected by a topological method.

Intrinsically bent DNA sequences have been implicated in the activation of transcription by promoting juxtaposition of DNA sequences near the terminal loop of a superhelical domain. We have developed a novel topological assay for DNA looping based on lambda integrative recombination to study the effects of intrinsically bent DNA sequences on the tertiary structure of negatively supercoiled DNA. Remarkably, the localization of adenosine-tract (A-tract) sequences in the terminal loop of a supercoiled plasmid is independent of the extent of intrinsic bending. The results suggest that A-tract-containing sequences have other properties that organize the structure of superhelical domains apart from intrinsic bending and may explain the lack of conservation in the degree of A-tract-dependent bending among DNA sequences located upstream of bacterial promoters.

Anomalous rapid electrophoretic mobility of DNA containing triplet repeats associated with human disease genes.

Eight human genetic diseases have been associated with the expansion of CTG or CGG triplet repeats. The molecular etiology behind expansion is unknown but may involve participation of an unusual DNA structure in replication, repair, or recombination. We show that DNA fragments containing CTG triplet repeats derived from the human myotonic dystrophy gene migrate up to 20% faster than expected in nondenaturing polyacrylamide gels, suggesting the presence of an unusual DNA helix structure within the CTG triplet repeats. The anomalous migration is dependent upon the number of triplet repeats, the length of the flanking DNA, and the percentage and temperature of the polyacrylamide. The effect could be reduced by the addition of actinomycin D. Applying a reptation model for electrophoresis, the results are consistent with a 20% increase in persistence length of the DNA. PCR products containing CTG or CGG repeats from the spinocerebellar ataxia type I gene (SCA1) or the fragile X FMR1 gene, respectively, also showed higher electrophoretic mobility. These are the first sequences of defined length for which a dramatic increase in mobility can be attributed to sequence-dependent structural elements in DNA.

Analysis of the structure of dimeric DNA catenanes by electron microscopy.

We analyzed the structure of open-circular and supercoiled dimeric DNA catenanes generated by site-specific recombination in vitro. Electron microscopy of open-circular catenanes shows that the number of duplex crossings in a plane is a linear function of the number of catenane interlinks (Ca/2), and that the length of the catenane axis is constant, independent of Ca. These relationships are similar to those observed with supercoiled DNA. Statistical analyses reveal, however, that the conformations of the individual rings of the catenanes are similar to those of unlinked circles. The distribution of distances between randomly chosen points on separate rings depends strongly on Ca and is consistent with a sharp decrease in the center-of-mass separation between rings with increasing Ca. Singly linked supercoiled catenanes are seen by microscopy to be linked predominantly through terminal loops in the respective superhelices. The observations suggest that chain entropy is a major factor determining the conformation of DNA catenanes.

Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.

Theories of pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) is one of the key technological advances of the past ten years that has made the mapping of genomes of whole organisms possible. In conventional electrophoreis, the mobility of DNA at almost any practical value of the field strength is essentially independent of mol wt above approx 30 kbp. Therefore, large (≥250 kbp) fragments of DNA required for mapping the genomes of entire organisms could not be separated prior to the introduction of these new electrophoresis techniques.

Understanding the anomalous electrophoresis of bent DNA molecules: a reptation model.

In polyacrylamide gel electrophoresis, the retardation of DNA molecules containing regions of intrinsic curvature can be explained by a novel reptation model that includes the elastic free energy of the DNA chain. Computer simulations based on this model give results that reproduce the dependence of anomalous mobility on gel concentration, which is quantified by new experimental data on the mobilities of circularly permuted isomers of kinetoplast DNA fragments. Fitting of the data required allowing for the elasticity of the gel.

Separations of open-circular DNA using pulsed-field electrophoresis.

The effect of high electric fields on the gel-electrophoretic mobility of open-circular DNA in agarose differs dramatically from that on linear molecules of the same molecular weight. At high fields, sufficiently large circular forms are prevented from migrating into the gel whereas linear molecules and smaller circular DNAs migrate normally. This effect is strongly field dependent, affecting circular molecules of decreasing size with increasing field strength. We have studied this effect with a series of plasmid DNAs ranging from 2.9 to 56 kilobase pairs using continuous and reversing-pulse electric fields. Application of reversing pulses abolishes the effect under certain conditions and supports the model for the gel electrophoresis of open-circular DNA where circular forms are trapped by engaging the free end of an agarose gel fiber.

Bending and flexibility of kinetoplast DNA.

We have evaluated the extent of bending at an anomalous locus in DNA restriction fragments from the kinetoplast body of Leishmania tarentolae using transient electric dichroism to measure the rate of rotational diffusion of DNA fragments in solution. We compare the rate of rotational diffusion of two fragments identical in sequence except for circular permutation, which places the bend near the center in one case and near one end of the molecule in the other. Hydrodynamic theory was used to conclude that the observed 20% difference in rotational relaxation times is a consequence of an overall average bending angle of 84 +/- 6 degrees between the end segments of the fragment that contains the bending locus near its center. If it is assumed that bending results from structural dislocations at the junctions between oligo(dA).oligo(dT) tracts and adjacent segments of B DNA, a bend angle of 9 +/- 0.5 degrees at each junction is required to explain the observations. The extent of bending is little affected by ionic conditions and is weakly dependent on temperature. Comparison of one of the anomalous fragments with an electrophoretically normal control fragment leads to the conclusion that they differ measurably in apparent stiffness, consistent with a significantly increased persistence length or contour length in the kinetoplast fragments.

Ring closure probabilities for DNA fragments by Monte Carlo simulation.

The rate of ligation of DNA molecules into circular forms depends on the ring closure probability, commonly called the j-factor, which is a sensitive measure of the extent to which thermal fluctuations contribute to bending and twisting of DNA molecules in solution. We present a theoretical treatment of the cyclization equilibria of DNA that employs a special Monte Carlo method for generating large ensembles of model DNA chains. Using this method, the chain length dependence of the j-factor was calculated for molecules. in the size range 250 to 2000 base-pairs. The Monte Carlo results are compared with recent analytical theory and experimental data. We show that a value of 475 A for the persistence length of DNA, close to values measured by a number of other methods, is in excellent agreement with the cyclization results. Preliminary applications of the Monte Carlo method to the problem of systematically bent DNA molecules are presented. The calculated j-factor is shown to be very sensitive to the amount of bending in these fragments. This fact suggests that ligase closure measurements of systematically bent DNA molecules should be a useful method for studying sequence-directed bending in DNA.

Topological distributions and the torsional rigidity of DNA. A Monte Carlo study of DNA circles.

Distributions of the linking number of circular DNA molecules, defined as the sum of twist and the writhing number, are obtained by Monte Carlo simulations of small, randomly closed DNA circles. We estimate the relative contributions of fluctuations in twist and writhe to the linking number distribution, as functions of DNA size. Published experimental data on topoisomer distributions in circular DNA molecules are interpreted to estimate the torsional rigidity of DNA in solution. We show that ignoring the writhe component of the linking number distribution, even for DNA circles as small as 250 base-pairs, leads to an underestimate for the torsional stiffness of the double helix. The value of the torsional modulus obtained from this analysis, C = 3.4 X 10(-19) erg cm, is from 10 to 40% larger than that estimated by others and more than twice as large as the values obtained from fluorescence depolarization or other time-resolved spectroscopic measurements. We also develop further the theoretical treatment of ring closure probabilities for DNA described in the previous article. It is shown that the torsional part of the ring closure probability, phi 0,1 (tau 0) is a periodic function of DNA length that contributes strongly to the ring closure probability for short chains but makes negligible contributions for chains over 1000 base-pairs in length.

A computer graphics study of sequence-directed bending in DNA.

We present theoretical results to account for the unusual physical properties of a 423 bp DNA restriction fragment isolated from the kinetoplast of the trypanosomatid Leishmania tarentolae. This fragment has an anomalously low electrophoretic mobility in polyacrylamide gels and a rotational relaxation time smaller than that of normally-behaved control fragments of the same molecular weight. Our earlier work (Proc. Natl. Acad. Sci. USA 79, 7664, 1982) has attributed these anomalies to the highly periodic distribution of the dinucleotide ApA in the DNA sequence. As originally proposed by Trifonov and Sussman (Proc. Natl. Acad. Sci. USA 77, 3816, 1980) local features of the DNA structure such as a small bend at ApA, if repeated with the periodicity of the helix, will cause systematic bending of the molecule. Computer graphics representations of DNA chain trajectories are presented for different structural models. It is shown that the structural model of Calladine (J. Mol. Biol. 161, 343, 1982) which is based on crystallographic data, is unsuccessful in predicting the systematic bending of DNA in solution.

Bent helical structure in kinetoplast DNA.

We have investigated the unusual physical properties of a restriction fragment of Leishmania tarentolae kinetoplast DNA. A gel-purified fragment comprising slightly more than half of a minicircle was determined by Maxam-Gilbert sequence determination to be 490 base pairs (bp) in length. This fragment has dramatically anomalous electrophoretic behavior; it has an apparent size of 450 bp on a 1% agarose gel but migrates as 1,380 bp on a 12% polyacrylamide gel. However, in gel filtration on Sephacryl S-500, the fragment elutes with an apparent size of 375 bp. Finally, it behaves anomalously in electric dichroism experiments. Field-free rotational relaxation times from transient electric dichroism studies are highly sensitive to effective molecular dimensions. The rotational relaxation time of the kinetoplast fragment is smaller than that of a 309-bp control fragment from pBR322. Because rigorous control experiments rule out the possibility that this fragment is modified, these anomalous properties must be dictated by the sequence itself. Fragment behavior indicates that it has an unusually compact configuration; we propose that this molecule contains a region of systematically bent B-DNA. This model accounts for the fragment's difficulty in snaking through the pores of a polyacrylamide gel, its ease in diffusing into Sephacryl beads, and its smaller rotational relaxation time. Bending of this molecule may be caused by periodicities in the DNA sequence.