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S Diekmann

Publications and source records attributed to S Diekmann.

At least 73 records · Page 4Linked to original sources
PubMed1989

Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli.

Can a transcriptional activator known to bend DNA be functionally replaced by a sequence-directed bend in Escherichia coli? To investigate this question, a partially truncated promoter was used, deleted of its -35 region and of its CRP binding site, leaving only two Pribnow boxes as functional elements. Synthetic and naturally occurring curved DNA sequences introduced upstream from these elements could restore transcription at either one of the two natural starts. Some of these hybrid promoters turned out to be more efficient than the CRP activated wild-type gal promoter in vivo. Control experiments performed with very similar sequences devoid of any curvature produced weak promoters only. Minimal changes in the location of the centre of curvature or perturbation in the amount of curvature strongly affected the level of expression. No significant stimulation of transcription could be detected in vitro. Furthermore, both gal P1 and P2 starts could be activated in vivo but also in vitro via a properly positioned CRP binding site. This partial analogy suggests that bending induced by the cAMP-CRP complex upon binding to its site may be biologically relevant to the mechanism of transcriptional activation.

Base Sequence↗
PubMed1989

Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules.

The four-way junction between DNA helices is the central intermediate in recombination, and the manner of its interaction with resolvase enzymes can determine the genetic outcome of the process. A knowledge of its structure is a prerequisite to understanding the interaction with proteins, and there has been recent progress. Here we use fluorescence energy transfer to determine the relative distances between the ends of a small DNA junction, and hence the path of the strands. Our results are consistent with the geometry of an 'X'. The interconnected helices are juxtaposed so that the continuous strands of each helix generate an antiparallel alignment, and the two interchanged strands do not cross at the centre. The acute angle of the X structure is defined by a right-handed rotation of the helical axes about the axis perpendicular to the X plane, as viewed from the centre of the X.

Base Sequence↗
PubMed1988

The structure of the Holliday junction, and its resolution.

The Holliday (four-way) junction is a critical intermediate in homologous genetic recombination. We have studied the structure of a series of four-way junctions, constructed by hybridization of four 80 nucleotide synthetic oligonucleotides. These molecules migrate anomalously slowly in gel electrophoresis. Each arm of any junction could be selectively shortened by cleavage at a unique restriction site, and we have studied the relative gel mobilities of species in which two arms were cleaved. The pattern of fragments observed argues strongly for a structure with two-fold symmetry, based on an X shape, the long arms of which are made from pairwise colinear association of helical arms. The choice of partners is governed by the base sequence at the junction, allowing a potential isomerization between equivalent structural forms. Resolvase enzymes can distinguish between these structures, and the resolution products are determined by the structure adopted, i.e., by the sequence at the junction. In the absence of cations, the helical arms of the junction are fully extended in a square configuration, and unstacking results in junction thymines becoming reactive to osmium tetroxide.

Base Sequence↗
PubMed1988

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction.

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.

Base Sequence↗
PubMed1988

Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences.

Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.

Base Sequence↗
PubMed1987

DNA methylation can enhance or induce DNA curvature.

Oligomers of different palindromic sequences (EcoRI, BamHI, and ClaI linkers) were ligated to form distributions of multimers. These long ligation ladders were methylated using corresponding methylases. The migration of the unmethylated as well as the methylated multimer distributions was analysed in 10% polyacrylamide gels. The migration anomaly of these sequences is interpreted in terms of the curvature of the DNA helix axis. The double-stranded oligomer dCGGAATTCCG is considerably curved in its unmodified form. Its curvature is strongly enhanced when the central dAs are methylated. This result is predicted by a model for DNA curvature. Multimers of the closely related sequence dCGGGATCCCG are straight. When methylated at the central dAs or at the most central dCs, a small curvature of the helix axis is induced. The double-stranded oligomer dCCATCGATGG is straight in its unmodified form as well as when it is methylated. Thus, DNA curvature can be induced or enhanced by methylation. However, DNA methylation at palindromic sequences seems not always to influence the linear path of the DNA helix axis.

Base Sequence↗
PubMed1987

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs.

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.

Animals↗
PubMed1987

The anomalous gel migration of a stable cruciform: temperature and salt dependence, and some comparisons with curved DNA.

We have made an analysis of the gel electrophoretic properties of a pseudo-cruciform fragment, a linear DNA molecule containing a stable cruciform. The migration of this construct was analysed in polyacrylamide gels at a various temperatures in the range 5 degrees to 55 degrees C, and in the presence of NaCl, MgCl2 or ethidium bromide. The magnitude of the anomalous migration (retardation) was almost temperature independent up to 40 degrees C, but decreased strongly beyond this point, extrapolating to normal migration at 70 degrees C. Addition of salts reduced the anomaly. This took the form of a continuous reduction in anomalous migration with the addition of NaCl up to 60 mM, while with MgCl2 there was a sharp reduction in the anomaly to a constant value which is reached by 10 mM. Under these conditions, moreover, the migration of the fragment became almost temperature-independent over the entire range. These results have been interpreted to reflect the influence of ion binding at the four-way junction on the relative disposition of the cruciform arms. The detailed electrophoretic properties of the pseudo-cruciform are in marked contrast to those of sequence-directed curved DNA fragments. In particular, the response to the addition of 1 microgram/ml ethidium bromide offers a convenient method for distinguishing between anomalous retardation arising from curvature (greatly reduced anomaly) or a cruciform junction (enhanced anomaly).

Coliphages↗
PubMed1987

Electro-optical analysis of 'curved' DNA fragments.

The structure of some 'short' DNA fragments with 106-108 base-pairs and of a 'long' kinetoplast DNA fragment with 419 base-pairs has been analyzed by electro-optical procedures. According to their electrophoretic mobilities and circularization probabilities, it was concluded that two of our short fragments with clusters of four and five and six adenosines phased at the period of the double helix are inherently curved with an approximate curvature around 200 degrees. The dichroism decay curves of our short fragments exhibited two processes. A fast one with time constants of approx. 100 ns is attributed to bending; the bending amplitudes observed for the fragments with dA4 and dA5/6 clusters are slightly higher (23 and 29%, respectively) than those observed for control fragments (17-20%). The second process reflects the overall rotational diffusion of the whole fragments and shows some variation with the DNA sequence, but on average the rotation of fragments with dA4 and dA5/6 clusters corresponds to that observed for standard DNA. Since the rotational diffusion coefficients are very strongly dependent on the effective hydrodynamic lengths, we must conclude that the effective lengths of our fragments, including the 'curved' ones, are very similar under the conditions of our experiments. The rotation time constant for the long kinetoplast DNA is also rather close to those observed for the usual DNA fragments of corresponding length. One way to resolve the conflict of our results with conclusions obtained from other investigations would invoke the assumption that the curved fragments are not 'elastic'. According to this hypothesis, electric field pulses would stretch the curved fragments to an almost straight form and the stretched DNA would return to its equilibrium state with a time constant longer than the rotation time constant.

Base Sequence↗
PubMed1987

Temperature and salt dependence of the gel migration anomaly of curved DNA fragments.

A series of oligonucleotides of different sequences have been cloned to study DNA curvature. Several DNA fragments containing these oligonucleotides in various numbers of repeats were analyzed in 10% polyacrylamide gels. A strong gel migration anomaly was found for dA4 sequences; a comparably very small but clearly detectable anomaly was observed for dA3 (both in a repeat length of 10 base-pairs). The temperature and salt (NaCl, MgCl2) dependence of the gel migration anomaly of these DNA fragments was measured. While a similar behaviour of all sequences is observed for the addition of NaCl, the temperature and MgCl2 dependence of the anomaly varies with the oligonucleotide sequence. These data are interpreted in terms of local DNA structure changes induced by changes in the temperature and the MgCl2 concentration which affect the planarity of the curved DNA fragments.

Base Sequence↗
PubMed1987

Molecular mechanics calculations of dA12.dT12 and of the curved molecule d(GCTCGAAAAA)4.d(TTTTTCGAGC)4.

Using the AMBER software package (Weiner and Kollman 1981) substantially modified for electrostatic contributions, the structural energies of the double-stranded oligonucleotides dA12.dT12 and d(GCTCGAAAAA)4.d(TTTTTCGAGC)4 were minimized. Using various starting structures for the molecule dA12.dT12, one final structure is obtained which possesses the experimentally determined properties of poly(dA).poly(dT). This structure is an A-form-B-form-hybrid structure similar to that of Arnott et al. (1983). The dA-strand is similar to an A-form while the dT-strand is similar to normal B-form. This structure and separately optimized B-form sequence stretches were used to construct the double-stranded fragment d(GCTCGAAAAA)4 which again was optimized. This sequence, when imbedded in a DNA fragment as contiguous repeats, shows a gel migration anomaly which has been interpreted as stable curvature of the DNA (Diekmann 1986). The calculated structure of this sequence indeed has a curved helix axis and is discussed as a model for curved DNA. A theoretical formalism is presented which allows one to calculate the structural parameters of any nucleic acid double helix in two different geometrical representations. This formalism is used to determine the parameters of the base-pair orientations of the curved structure in terms of wedge as well as cylindrical parameters. In the structural model presented here, the curvature of the helix axis results from an alternation of two different DNA structures in which the base-pairs possess different angles with the helix axis ('cylinder tilt'). Resulting from geometric restraints, a negative cylinder tilt angle correlates strongly with the closing of the minor groove ('wedge roll'). The blocks with different structure are not exactly coincident with the dA5-blocks and the B-DNA stretches. Within the dA5 block, base-pair tilt and wedge roll adopt large values which proceed into the 3' flanking B-DNA sequence by about one base-pair. These properties of the structure calculated here are discussed in terms of different models explaining DNA curvature.

DNA↗
PubMed1987

The influence of exocyclic substituents of purine bases on DNA curvature.

Complementary oligonucleotides with 5' overhanging deoxyguanosine or deoxycytidine stretches, respectively, of the general form 5'-d(GGGCAARAAC).5'-d(CCCGTTYTTG), where R represents the bases adenine (A), hypoxanthine (base of inosine nucleoside, I), purine (R), 2-aminopurine (n2R), or 2,6-diaminopurine (n2,6(2)R) and where Y represents the pyrimidine bases thymine (T) or cytosine (C), have been chemically synthesized. After hybridization of complementary fragments, they were ligated to form multimers and analyzed by polyacrylamide gel electrophoresis. Anomalous gel migration was observed for the sequences 5'-d(AARAA) when the R.Y base pair was dA.dT, dI.dC, or dR.dT. All of these base pairs lack at least the amino group at position 2 of the purine base. The degree of anomalous gel migration was also related to the substituent at position 6 of the purine base. An amino group at position 6 was more effective than a carbonyl or a hydrogen in inducing anomalous gel migration. Additionally, the fragments 5'-d(GGGCAIAIAC).5'-d(CCCGTCTCTG), 5'-d(GGGCAIIIAC).5'-d(CCCGTCCCTG), and 5'-d(GGGCIIAIIC).5'-d(CCCGCCTCCG) were prepared in which increasing numbers of dA.dT base pairs are replaced by dI.dC base pairs. The degree of gel-migration anomaly of these sequences correlates with the number of dA.dT base pairs left in the five-base purine block. The data support the hypothesis that within the deoxyadenosine tracts, the base pairs fold into the minor groove at position 2 of the base to balance for the NH2 groups at position 6. This hypothesis explains the formation of a B'-form DNA structure for the deoxyadenosine tracts as well as DNA curvature.

DNA↗
PubMed1986

Sequence specificity of curved DNA.

Anomalously slow migration of DNA fragments on polyacrylamide gels is interpreted as resulting from curvature of the DNA fragment. Different models have been suggested to explain DNA curvature. In this work a number of DNA fragments were synthesized, cloned, and electrophoretically characterized to distinguish between these models. Strong anomaly of migration is found for sequence stretches (dA)n repeated in phase with the helix turn with n at least 4. For n smaller than 4 only negligible anomaly is observed. The results contradict the purine-clash hypothesis. The data can be explained by assuming longer stretches of As to be in a B'-form, and that tilt of this structure might be the reason for its curvature.

Base Sequence↗
PubMed1985

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.

Animals↗
PubMed1982

Orientation relaxation of DNA restriction fragments and the internal mobility of the double helix.

The orientation relaxation of 15 DNA restriction fragments (43-4361 base-pairs) is characterized by measurements of linear dichroism using high electric field pulses. The off-field relaxation of fragments of 84 base-pairs or less can be described by single exponentials, which are related to the transverse rotational diffusion of the helix. Fragments of 95 base-pairs or greater exhibit an additional fast component with time constants around 100 ns for fragments of approx. 100 base-pairs, increasing with chain length to about 700 ns for a fragment with 258 base-pairs. The amplitude of this process increases from virtually zero at low fields (approximately equal to 10 kV) to a substantial limit contribution at high fields. According to these results, we suggest that electric fields induce stretching of the DNA fragments from a weakly bent to a more straight form and that the fast component reflects the internal mobility of the DNA chain. The slow off-field components of the orientation are discussed in terms of different models. The data up to helix lengths of about 400 base-pairs can be described by the 'weakly bending rod' model from Hearst using 3.4 A rise per base-pair and 13 A axial radius of the helix. Both the weakly bending rod according to Hearst and the 'wormlike chain' according to Hagerman and Zimm provide a persistence length of 500 A. The on-field relaxation is slower than the corresponding off-field process at low field strengths, but the on-field process is accelerated substantially at high electric fields. These observations are compared with model calculations of Schwarz.

Base Composition↗
PubMed1982

Electric properties and structure of DNA-restriction fragments from measurements of the electric dichroism.

The electric dichroism of 17 homogeneous DNA fragments, ranging in size from 43 to 4362 base-pairs, has been analyzed in high electric fields. The orientation of the small fragments can be described in terms of an induced dipole moment, whereas the large fragments are oriented according to a constant dipole mechanism. In the intermediate size range, DNA orients according to an induced dipole mechanism at low field strengths and according to a constant dipole mechanism at high field strengths. From these observations we propose an orientation mechanism with a saturating induced dipole. The induced dipole observed at low field strengths is saturated at a field strength Eo within a transition range Em to give a constant dipole moment at high field strengths. These parameters together with the polarizability and the limit reduced dichroism are evaluated by a least-squares analysis of the experimental data. Eo and Em are found to decrease with increasing chain length from Eo approximately 40 kV/cm (Em approximately 14 kV/cm) at 65 base-pairs to 10 kV/cm (6 kV/cm) at 194 base-pairs. The polarizability is found to increase with the square of the chain length, whereas the saturated dipole increases with chain length N at low N and goes to a limit value at high N. The temperature dependence of the orientation parameters is found to be very small. The values obtained for the limit dichroism are between -1.0 and -1.3 for chain lengths between 60 and 1000 base-pairs, whereas values around -1.4 are observed at chain lengths greater than 1000 base-pairs. These data indicate that electric fields extend the contour of DNA strands at high chain lengths from a weakly bent to a more linear form. The variations of the limit dichroism observed for short fragments suggest sequence-dependent differences in the secondary structure of the helix. The experimental results are compared with numerical calculations based on simple polyelectrolyte models. For short fragments the magnitude of several electrochemical parameters can be adequately explained by a polarization of the ion cloud around the DNA molecules. However, these polyelectrolyte models do not adequately describe the observed chain length dependence of the orientation phenomena.

Base Composition↗
PubMed1982

Thresholds in field-induced reactions of linear biopolymers. Strong chain-length dependence of field effects in DNA.

We have analysed the field-induced conformation change of DNA by absorbance measurements at the magic angle. Conformation changes are observed when the electric field strength exceeds a clearly defined threshold value. The threshold values increase with increasing salt concentration and show a linear dependence upon the logarithm of the ionic strength. Measurements with homogeneous DNA samples of different chain lengths N show that the threshold increases with decreasing N; at a given ionic strength the threshold is a linear function of the logarithm of N. The threshold value observed for a circular DNA molecule with a chain length Nc fits to these data with an effective length Nc/2. This result indicates that the length of maximal extension is important for the field-induced reaction and suggests, together with the other results, that the field-induced reaction is mainly driven by a polarization of the ion atmosphere along the axis of DNA. Some data are also given for the dynamics of the reaction: at high electric field pulses the first step is a fast destacking and tilting of the bases followed by a slow unwinding process. For short pulses the reaction is almost completely reversible with a characteristic time constant of about 3 microseconds for the back reaction.

Animals↗