Intrinsic DNA Curvature Research Articles

Scanning tunneling investigation of DNA structures involved in gene regulation

Scanning tunneling microscopy (STM) observations made on pBR322 DNA are compared with methods known to give information on DNA intrinsic curvature: polyacrylamide gel retardation, electron microscopy and computer modeling. It is concluded that STM is a powerful method for studying DNA structural features. Moreover, it is the only method to reveal simultaneously nanometric scale detail and long-range information. Use of STM for analyzing fine DNA structural features in regions involved in gene regulation should make an important contribution to genetics.

Effects of DNA topology in the interaction with histone octamers and DNA topoisomerase I

Several simple proteins and complex protein systems exist which do not recognize a defined sequence but - rather - a specific DNA conformation. We describe experiments and principles for two of these systems: nucleosomes and eukaryotic DNA topoisomerase I. Evidences are summarized that describe the effects of negative DNA supercoiling on nucleosome formation and the influence of DNA intrinsic curvature on their localization. The function of the DNA rotational information in nucleosome positioning and in the selection of multiple alternative positions on the same helical phase are described. This function suggests a novel genetic regulatory mechanism, based on nucleosome mobility and on the correlation between in vitro and in vivo positions. We observe that the same rules that determine the in vitro localization apply to the in vivo nucleosome positioning, as determined by a technique that relies on the use of nystatin and on the import of active enzymes in living yeast cells. The sensitivity of DNA topoisomerase I to the topological condition of the DNA substrate is reviewed and discussed taking into account recent experiments that describe the effect of the DNA tridimensional context on the reaction. These topics are discussed in the following order: (i) Proteins that look for a consensus DNA conformation; (ii) Nucleosomes; (iii) Negative supercoiling and nucleosomes; (iv) DNA curvature/bending and nucleosomes; (v) Multiple positioning; (vi) Multiple nucleosomes offer a contribution to the solution of the linking number paradox; (vii) Rotational versus; translational information; (viii) A regulatory mechanism; (ix) DNA topoisomerase; I; (x) DNA topoisomerase I and DNA supercoiling: a regulation by topological feedback; (xi) DNA topoisomerase I and DNA curvature; (xii) The in-and-out problem in the accessibility of DNA information; (xiii) The integrating function of the free energy of supercoiling.

CA/TG sequence at the 5' end of oligo(A)-tracts strongly modulates DNA curvature.

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAAC-CCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

Structural characterization of intrinsically curved AT-rich DNA sequences.

AT-rich DNA sequences other than AnTm tracts (n + m > or = 4) are known to be intrinsically curved. The AATAT-element constitutes one known example of these sequences. In this paper, the elucidation of the structural basis of the curvature induced by this sequence element was addressed. As judged by the patterns of cleavage by the hydroxyl radical and DNase I, the AATAT sequence shows a narrow minor groove. Furthermore, the 5' adenine residue of the AA dinucleotide contained within the sequence is hyperreactive to diethylpyrocarbonate. Similar structural properties are shown by several sequences inducing intrinsic DNA curvature, such as an A5-tract or the closely related ATAAT, AATATA and TAATAT sequences, which are also shown here to induce curvature. On the other hand, other related sequences, such as TATAA and ATATA, that do not induce curvature, show different structural characteristics.

Role of the Upstream Region Containing an Intrinsic DNA Curvature in the Negative Regulation of the Phospholipase C Gene of Clostridium perfringens

The phospholipase C (alpha-toxin) gene (plc) of Clostridium perfringens was cloned into pUC19 and the effects of the upstream regions on expression of the plc gene were examined in Escherichia coli JM109. When the 0.7-kb region just upstream of the putative -35 site of the gene was deleted, production of phospholipase C increased approximately 10-fold. Northern blot hybridization analysis of the plc transcript showed that the upstream region inhibited transcription from the plc promoter. Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)5-6 tracts between positions -66 and -40 of the plc gene. A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the plc promoter is a locus of sequence directed DNA-bending. Nested deletions of the upstream region were created from its 5' end by exonuclease III and the effects of deletions on the expression of the plc gene were examined. When the 77-bp fragment containing the two (dA)5-6 tracts were deleted, phospholipase C production increased markedly. These results indicate that the intrinsic DNA curvature upstream of the plc promoter is involved in the negative regulation of the plc gene transcription.

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.

Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles.

The principal sequence feature responsible for intrinsic DNA curvature is generally assumed to be runs of adenines. However, according to the wedge model of DNA curvature, each dinucleotide step is associated with a characteristic deflection of the local helix axis. Thus, an important test of a more general view of sequence-dependent DNA curvature is whether sequence elements other than A-A cause the DNA axis to deflect. To address this question, we have applied the wedge model to a large body of experimental data. The axial path of DNA can be described at each step by three Eulerian angles: the helical twist, the deflection angle (wedge angle), and the direction of the deflection. Circularization and gel electrophoretic mobility data on 54 synthetic DNA fragments, both from other laboratories and from our own, were used to compare the theoretical predictions of the wedge model with experiment. By minimizing misfit between calculated and observed DNA curvature, we have found that the stacks AG/CT, CG/CG, GA/TC, and GC/GC, in addition to AA/TT, have large wedge values. We have also synthesized seven sequences without AA/TT elements but with these other wedges correctly phased to cause appreciable predicted curvature. All appear curved as demonstrated by anomalous gel mobilities. The full set of 16 roll and tilt wedge angles is estimated and, together with the known 10 helical twists, these allow prediction of the general sequence-dependent trajectory of the DNA axis.

The intrinsic curvature of DNA in solution

We propose a detailed quantitative scheme for explaining the anomalous electrophoretic mobility in polyacrylamide gels of repeating sequence DNA. We assume that such DNA adopts a superhelical configuration in these circumstances, and migrates less quickly than straight DNA of the same length because it can only pass through larger holes. The retardation is maximal when the length of the DNA reaches one superhelical turn, but is less for shorter pieces. We attribute the curvature of the superhelix to different angles of roll at each kind of dinucleotide step, i.e. an opening up of an angle by an increased separation on the minor-groove side. The main effect is due to a difference of about 3 ° in roll values between AA TT and other steps, together with a difference of about 1 ° in the angle of helical twist: we deduce these values explicitly from some of the available data on gelrunning. The scheme involves a simple calculation of the superhelical parameters for any given repeating sequence, and it gives a good correlation with all of the available data. We argue that these same base-step angular parameters are also consistent with observations from X-ray diffraction of crystallized oligomers, and particularly with the recent data on CGCA 6GCG from Nelson et al. We are concerned here with the intrinsic curvature of unconstrained DNA, as distinct from the curvature of DNA in association with protein molecules; and this paper represents a first attempt at an absolute determination.