Visualization of the bent helix in kinetoplast DNA by electron microscopy

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.     

HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.     

Determination of the extent of DNA bending by an adenine-thymine tract

We determined the magnitude of the bend induced in DNA by an adenine-thymine tract by measuring the rate of cyclization of DNA oligonucleotides containing phased A tracts. A series of linear multimers with 2-bp single-stranded ends, in which the (A.T)6 tracts are separated by CG2-3C sequences and are positioned 10 and 11 bp apart alternately, were prepared from 21 bp long synthetic duplexed deoxyoligonucleotides. The cyclization rates of the multimers (105-210 bp) and the bimolecular association rate of the 84 bp long multimer were measured in the presence of DNA ligase. From the rate constants of the cyclization and bimolecular association reactions, ring closure probabilities were obtained for the multimers. The systematically bent molecules were simulated by Monte Carlo methods, and the ring closure probabilities were calculated for a given set of junction bend angles. By comparing the calculated values of ring closure probabilities to experimental values and adjusting the junction bend angles to fit experimental values, the extent of bending at the junctions (or the extent of bending for an adenine tract) was determined. We conclude that an A6 tract bends the DNA helix by 17-21 degrees.     

DNA bending induced by high mobility group proteins studied by fluorescence resonance energy transfer.

The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures. DNA binding also induces a substantial bend. Using fluorescence resonance energy transfer (FRET), we have determined the changes in the end-to-end distance consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges. The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively. The former value contrasts with an inferred value of 150 degrees reported by Heyduk et al. (1) for the bend induced by a single domain. We also observe that the induced bend angle is unaffected by the presence of the C-terminal acidic region. The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees). We suggest this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif.     

Conservation and continuity of periodic bent DNA in genomic rearrangements between the c-myc and immunoglobulin heavy chain mu loci.

Periodic bent DNA was mapped in the human c- myc and immunoglobulin heavy chain mu (Ig mu) loci. A total of 12 DNA bend sites in the c- myc gene and 11 sites in the Ig mu locus were aligned at average intervals of 694.2 +/- 281.4 and 654.5 +/- 222.7 bp respectively. Although some of the bend sites retained the distance of 700 bp, their periodicity was disturbed at several locations, including the exons of the c- myc gene and the enhancer element present in the Ig mu locus. Analysis of rearrangements that resulted in tumorigenesis of lymphocytes showed that the continuity of DNA bend sites was conserved in three lymphoma cell lines, Manca, BL22 and Ramos, suggesting that the genomic rearrangements gain stability by retaining their periodicity. This adds further evidence that the periodic bent DNA plays a crucial role in genomic structure.     

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB.     

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.     

Random cloning of bent DNA segments from Saccharomyces cerevisiae and primary characterization of their structures

Summary Until recently it was assumed that any short segment of DNA could be approximated as a straight rod. Many instances, however, have been reported in which the helical axis is curved. We have devised a simple method for selective identification of DNA segments containing a sequence-directed bend (curvature), by means of a two-dimensional polyacrylamide gel electrophoresis. In order to gain general insights into the structural features and the functional significance of sequence-directed bends, a bank of plasmids carrying bent DNA inserts from the Saccharomyces cerevisiae total genomic DNA was constructed. Primary characterizations of a set of bent DNA segments randomly cloned from S. cerevisiae are presented. One of the cloned DNA segments appears to be derived from a yeast plasmid, the 2 m circle DNA.     

Detection, sequence patterns and function of unusual DNA structures.

Unusual DNA structures were detected by an electrophoretic procedure in which DNA fragments were separated according to size on agarose gels and then by shape on polyacrylamide gels. Fragments from yeast centromeres migrated faster in polyacrylamide than predicted from their base composition and size and this property was attributed to a nonrandom distribution of oligomeric A tracts that exhibited minima at 10-11 base intervals. Fragments from seven loci in 107 kb of DNA migrated anomalously slow and these fragments contained blocks of A2-6 in a 10-11 base periodicity which is indicative of bent DNA. The most pronounced bent sequences were found within yeast ARS1 and centered at 245 and 240 bp from the left and right ends of the adenovirus genome. Each sequence is approximately 150 bp away from a replication origin and the adenovirus sequences are within 50 bp of enhancers. Nuclear matrix attachment sites, which are also adjacent to enhancers, contain sequences characteristic of bent DNA. These results suggest that bent structures reside at the base of DNA loops in chromosomes.     

Various elements of the structure of mini-ring kinetoplast DNA of Crithidia fasciculata

The nucleotide sequence of 1.4 kbp SmaI-fragment of minicircle DNA from kinetoplasts of Crithidia fasciculata has been determined and some sequence elements characterized. The sequence contains several oligo(dT)blocks located on the same strand in phase with a period of DNA helix turn, thus representing a "bent helix". Both sides of the bent helix region are flanked by sequences capable of forming a cloverleaf structure. There are also two direct 150 bp repeats located 180 degrees apart on the circular map of the molecule. Each repeat contains the sites of H-strand and L-strand replication origin. The specific stem-loop secondary structure may be folded by the nucleotide sequence within the origins region. The alignment of the sequence determined with two other C. fasciculata minicircle sequences spanning over the bent helix and the adjacent regions has indicated the presence of several conserved sequence blocks, one of them representing the sequence of the bend. The divergence of three sequences occurred mainly by small insertions-deletions. Several open reading frames were found, the largest of which being capable of coding for the approximately 200 amino acids polypeptide.     

Spontaneous sharp bending of double-stranded DNA

Sharply bent DNA is essential for gene regulation in prokaryotes and is a major feature of eukaryotic nucleosomes and viruses. The explanation normally given for these phenomena is that specific proteins sharply bend DNA by application of large forces, while the DNA follows despite its intrinsic inflexibility. Here we show that DNAs that are 94 bp in length-comparable to sharply looped DNAs in vivo-spontaneously bend into circles. Proteins can enhance the stability of such loops, but the loops occur spontaneously even in naked DNA. Random DNA sequences cyclize 10(2)-10(4) times more easily than predicted from current theories of DNA bending, while DNA sequences that position nucleosomes cyclize up to 10(5) times more easily. These unexpected results establish DNA as an active participant in the formation of looped regulatory complexes in vivo, and they point to a need for new theories of DNA bending.     

Modulation of intramolecular interactions in superhelical DNA by curved sequences: a Monte Carlo simulation study.

A Monte Carlo model for the generation of superhelical DNA structures at thermodynamic equilibrium (Klenin et al., 1991; Vologodskii et al., 1992) was modified to account for the presence of local curvature. Equilibrium ensembles of a 2700-bp DNA chain at linking number difference delta Lk = -15 were generated, with one or two permanent bends up to 120 degrees inserted at different positions. The computed structures were then analyzed with respect to the number and positions of the end loops of the interwound superhelix, and the intramolecular interaction probability of different segments of the DNA. We find that the superhelix structure is strongly organized by permanent bends. A DNA segment with a 30 degrees bend already has a significantly higher probability of being at the apex of a superhelix than the control, and for a 120 degrees bend the majority of DNAs have one end loop at the position of the bend. The entropy change due to the localization of a 120 permanent bend in the end loop is estimated to be -17 kJ mol-1 K-1. When two bends are inserted, the conformation of the superhelix is found to be strongly dependent on their relative positions: the straight interwound form dominates when the two bends are separated by 50% of the total DNA length, whereas the majority of the superhelices are in a branched conformation when the bends are separated by 33%. DNA segments in the vicinity of the permanent bend are strongly oriented with respect to each other.     

Topological measurement of an A-tract bend angle: variation of duplex winding

The rotational variant method of Lutter et al. was developed to measure the bend angle induced when a protein binds to DNA. To measure the intrinsic bend conferred by a sequence of six adenine bases (an A6 tract), the method was modified by relaxing at high temperature to remove the bend. We describe here an alternative approach that involves unwinding the duplex DNA between adjacent bends in plasmids containing tandemly repeated blocks of A-tracts. This method measures the topological difference contributed by adjacent bends when they are in two different rotational settings, and therefore does not require reference to a straight state. The interbend DNA was unwound by use of the intercalator chloroquine, or, alternatively, by raising the temperature in the relaxation reaction. The effect of this unwinding is to change the pitch of the superhelix of the tandem repeats from which the bend angle is measured. The result is a bend angle value that is consistent with that measured using the bend-straightening version of the method. This version offers several advantages that complement the conventional bent versus straight approach.