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Design and calibration of a semi-synthetic DNA phasing assay.
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PMID:  11095698     Owner:  NLM     Status:  MEDLINE    
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Electrophoretic assays of intrinsic DNA shape and shape changes induced by ligand binding are extremely useful because of their convenience and simplicity. The development of calibrations and empirical quantitative relationships permits highly accurate measurement of DNA shape using electrophoresis. Many conventional analyses employ the unidirectional ligation of short DNA duplexes. However, many oligonucleotides (typically more than 20) must often be synthesized for a single experiment. Additionally, the length of the DNA duplex can become limiting, preventing the analysis of certain DNA sequences. We now describe a semi-synthetic electrophoretic phasing method that offers several advantages, including a reduced number of required synthetic oligonucleotides, the ability to analyze longer DNA duplexes and a simplified approach for data analysis. We characterize semi-synthetic DNA probes in electrophoretic phasing assays by ligation of synthetic duplexes containing A(5) tracts between two longer restriction fragments. Upon electrophoresis, the gel mobility is strongly correlated with the predicted DNA curvature provided by the reference A(5) tracts. Having obtained this calibration, we show that the semi-synthetic phasing assay can be readily and economically applied to analyze DNA curvature induced by DNA charge modifications and DNA bending due to peptide binding.
Authors:
P R Hardwidge; J M Zimmerman; L J Maher
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Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Nucleic acids research     Volume:  28     ISSN:  1362-4962     ISO Abbreviation:  Nucleic Acids Res.     Publication Date:  2000 Dec 
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Created Date:  2000-12-04     Completed Date:  2001-01-25     Revised Date:  2009-11-19    
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Nlm Unique ID:  0411011     Medline TA:  Nucleic Acids Res     Country:  ENGLAND    
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Languages:  eng     Pagination:  E102     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905, USA.
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DNA / chemistry*,  genetics,  metabolism
DNA-Binding Proteins*
Electrophoresis / methods
Fungal Proteins / chemistry,  metabolism
Kinetics
Nucleic Acid Conformation*
Oligonucleotides / chemistry,  genetics
Protein Binding
Protein Kinases / chemistry,  metabolism
Saccharomyces cerevisiae Proteins*
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Chemical
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0/DNA-Binding Proteins; 0/Fungal Proteins; 0/Oligonucleotides; 0/Saccharomyces cerevisiae Proteins; 9007-49-2/DNA; EC 2.7.-/Protein Kinases
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Journal ID (nlm-ta): Nucleic Acids Res
ISSN: 0305-1048
ISSN: 1362-4962
Publisher: Oxford University Press, Oxford, UK
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Copyright ? 2000 Oxford University Press
Received Day: 3 Month: 8 Year: 2000
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Accepted Day: 1 Month: 10 Year: 2000
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PubMed Id: 11095698

Design and calibration of a semi-synthetic DNA phasing assay
Philip R. Hardwidge
Jeff M. Zimmerman
L. James Maher, IIIa
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905, USA
aTo whom correspondence should be addressed. Tel: +1 507 284 9041; Fax: +1 507 284 2053; Email: maher@mayo.edu

INTRODUCTION

The physical basis for both intrinsic DNA curvature and for curvature induced by proteins and chemical modifications is under intense study. DNA curvature and bending are analyzed using a number of methods. X-ray diffraction and NMR spectroscopy provide high resolution, but require large amounts of material. Spectroscopic tools such as FRET (1), LRET (2), TEB (3) and ultracentrifugation are also employed, but require expensive instrumentation. Enzyme-mediated cyclization (4,5) and differential protein binding to preformed DNA minicircles (6?8) have been used with small quantities of radiolabeled DNA.

Gel-based electrophoretic assays of DNA shape have also been extremely important because of their convenience and simplicity. Such methods include circular permutation assays, phasing assays and ligation ladder experiments (9). A rigorous physical theory of native gel electrophoresis through media such as polyacrylamide or agarose has not been developed. However, insightful calibrations and empirical quantitative relationships have allowed highly accurate measurement of DNA shape with electrophoresis.

Conventional ?comparative electrophoresis? analyses often employ the unidirectional ligation of short DNA duplexes (reviewed in 9). Duplexes constituting integral numbers of helical turns of DNA are chosen to contain variable spacer regions and intrinsically curved A5?6 tract sequences, so that the resulting ligation products allow the estimation of DNA bend angles following electrophoresis (9,10). Several duplex lengths are first studied to verify helical repeat parameters. Although useful and highly refined, this approach suffers from the requirement that a large number of oligonucleotides (typically more than 20) be synthesized for a single experiment to analyze properly DNA curvature in a particular sequence context (9,11,12). The length of the DNA duplex can become limiting, preventing the analysis of certain DNA sequences. We now describe a semi-synthetic electrophoretic phasing method that offers several advantages, including a reduced number of required synthetic oligonucleotides and a simpler approach for data analysis.

We first characterize semi-synthetic DNA probes in phasing assays by ligation of synthetic duplexes containing intrinsically curved DNA sequences between two longer probe arms prepared by recombinant techniques. Upon electrophoresis, the apparent gel mobility is correlated with the predicted DNA curvature provided by reference A5 tracts. Having obtained this calibration, we show that the semi-synthetic phasing assay is readily and economically applied to analyze DNA curvature induced by DNA charge modifications and DNA bending induced by proteins.


MATERIALS AND METHODS
Oligonucleotides

Unmodified oligonucleotides were prepared by standard phosphoramidite chemistry. All oligomers were purified by denaturing 20% polyacrylamide gel electrophoresis, eluted overnight from diced gel slices and desalted using C18 reverse phase cartridges. Oligonucleotide concentrations were determined at 260?nm using appropriate nearest neighbor molar extinction coefficients (M?1 cm?1) as described (13). Oligonucleotides containing methylphosphonate substitutions were synthesized and deprotected as described (14,15). In some cases oligonucleotides were characterized by electrospray ionization mass spectrometry (16).

PCR design

The plasmid pDP-AP-1-21 (17) was used as a PCR template for construction of the left and right probe arms. PCR primers for amplification of the left arm were: 5?-GCGCGA2GACT2CACGCGTAGA and 5?-CGCG2A2GAC2TCGAT2C2ATG. Top strand PCR primers for amplification of the five right arms were: R-A, 5?-GA2GACATGCTCAT2CTGCA5CG3C; R-B, 5?-GA2GACATGCTCATC2TCTGCA5CG3C; R-C, 5?-GA2GACATGCTCG2ATC2TCTGCA5CG3C; R-D, 5?-GA2GACATGCTC2G3ATC2TCTGCA5CG3C; R-E, 5?-GA2ACATGCTC2GCG3ATC2TCTGCA5CG3C. The bottom strand primer for all right arms was 5?-GCGCGA2GAC2TG2ATATCT3A3C. PCR of the left arm was performed using Taq DNA polymerase for five cycles of 94?C for 1 min, 27?C for 1 min and 72?C for 1 min, followed by 20 cycles of 94?C for 1 min, 49?C for 1 min and 72?C for 1 min. PCR of the right arms was performed for five cycles of 94?C for 1 min, 39?C for 1?min and 72?C for 1 min, followed by 20 cycles of 94?C for 1?min, 53?C for 1 min and 72?C for 1 min. PCR products were purified by electrophoresis through 2% agarose gels, followed by elution using Freeze n? Squeeze columns (Bio-Rad) as directed by the manufacturer.

Cloning of PCR products

Purified PCR products were ligated into the pGem-T vector (Promega). Putative clones were screened by digestion with either PvuII or SpeI and sequenced with the primer 5?-TA2TACGACTCACTATAG3. Each plasmid was digested with BbsI to release the desired left and right arms. PCR products were purified by electrophoresis through 2% agarose gels, followed by elution using Freeze n? Squeeze columns. The components were resuspended at a concentration of ?10?nM.

Design of duplex inserts

Oligonucleotides (36 nt) were purified by electrophoresis through 10% acrylamide gels containing 7.5 M urea. Oligonucleotides were end-labeled with [?-32P]ATP and T4 polynucleotide kinase, annealed to the appropriate complementary strand and resuspended at a concentration of ?10 nM.

Trimolecular ligations

Equimolar amounts (?1 nM final concentration) of the left arm, right arm and duplex inserts were ligated at room temperature for 2 h, using 400 U T4 DNA ligase (New England Biolabs) in a 10 ?l reaction. Ligations were terminated by the addition of EDTA to a final concentration of 50 mM.

Confirmation of ligation product trimolecularity

To confirm that ligation products were trimolecular, a subset of the ligation products were resolved on 5% preparative native polyacrylamide (1:29 bis-acrylamide:acrylamide) gels in 0.5? TBE buffer, at 10 V cm?1 at 22?C. After elution from the gel, purified ligation products were cloned into the pGem-T Easy vector (Promega) via installation of 3?-adenosine overhangs by incubation with Taq DNA polymerase (Gibco) and dATP.

Quantitative analyses of phasing probe mobilities

Ligation products (?240 bp) were resolved on 8% native polyacrylamide (1:29 bis-acrylamide:acrylamide) gels in 0.5? TBE buffer, at 10 V cm?1 at 22?C for 5 h. Mobilities of trimolecular products were measured (mm) and normalized to the average mobility of each group of five ligation products sharing the same duplex insert, but containing the five different right arms, according to:

?rel = ?/?avg1

where ?rel is the relative mobility, ? is the mobility of each ligation product and ?avg is the average mobility of a group of probes sharing the same duplex insert.

The value of ?rel was plotted against the spacing (in bp) between the double-stranded region of the duplex insert and the center of the 5? A5 tract in R-A to R-E and then fitted to the phasing function:

?rel = (APH/2){cos[2?(S ? ST)/PPH]} + 1 2

where APH is the amplitude of the phasing function, S is the normalized spacer length, ST is the trans spacer length and PPH is the phasing period (set at 10.5 bp per turn) (18). The value of APH estimated from curve fitting is related to the magnitude of curvature in the synthetic insert. The value of ST estimated from curve fitting allows evaluation of the direction of curvature relative to the phased A5 tract array intrinsic to the right arm.

Protein-mediated DNA bending assays

Charge variants of the DNA-binding domain of recombinant yeast GCN4 were expressed and purified to near homogeneity over Ni?NTA agarose columns (P.R.Hardwidge and L.J.Maher, unpublished results). Following trimolecular ligations (see above) the reaction was supplemented with 20 ng/?l poly(dI?dC) and sufficient peptide (?50 nM) to bind ?50% of the probe. Reactions were incubated for an additional 30 min at room temperature. DNA?protein complexes were resolved and quantitated as described above.


RESULTS AND DISCUSSION
Experimental design

Figure 1 illustrates the design of our semi-synthetic phasing assay. We designed ?260 bp linear DNA probes composed of three components. (i) The ?100 bp left arm is prepared as a linear DNA duplex PCR product (Fig. 1, L). Type IIS restriction endonuclease digestion of the PCR product creates a 5?-CGAT overhang on the bottom strand that can be utilized in ligations. This invariant left arm is prepared in bulk for use in all probes in this system. (ii) The ?120 bp right arm is prepared as a series of five different PCR products, each containing a region of constant curvature (phased A5 tracts) separated by a variable spacer from the left end of the arm (Fig. 1, R-A to R-E). Type IIS restriction endonuclease digestion of each PCR product creates a 5?-GCTC overhang that can be utilized in ligations. This 5? overhang is designed not to anneal to the 3? overhang of the left arm. (iii) Between these two PCR-generated probe arms is ligated the synthetic DNA duplex to be studied (Fig. 1, I). This duplex may contain chemical modifications and can be sufficiently long to accommodate additional intrinsically curved sequences and protein binding sites.

L and R-A to R-E were generated by PCR from plasmid pDP-AP-1-21 (17). PCR products containing appropriately placed internal BbsI sites were cloned into a pGem-T vector and the indicated left (L) and right arms (R-A to R-E) were released via BbsI digestion. BbsI is a type IIS restriction enzyme. This family of enzymes recognizes a specific DNA sequence, but makes staggered cuts at fixed distances from the recognition site. This property was advantageous in our design because it allowed the construction of molecular termini that were not self-complementary and that would anneal only to the appropriate end of the synthetic duplex insert. Synthetic duplex DNA inserts could thus be engineered to promote trimolecular ligations without competition from any self-dimerization reactions. Sequences were chosen so that the ligation products would be ?260 bp in length. We chose this design based on previous studies favoring phasing probes with long flanking sequences and loci of curvature separated by short spacers (19).

This trimolecular ligation scheme creates a series of five different probes. The length of DNA between the left terminus and the phased A5 tracts (providing ?54? of intrinsic curvature) in R-A to R-E differs for each insert (Fig. 1). As a result, there are five distinct helical phasings between elements in the synthetic insert (I) and the phased A5 tracts in R-A to R-E. This design allows quantitative analysis of the shape of the DNA in the synthetic insert.

After establishing optimal ligation conditions, we confirmed the trimolecularity of ligation products. Synthetic duplex insert 1 (Fig. 2) was ligated between L and each of the five right arms R-A to R-E. The resulting ligation products were purified and subsequently modified by installation of 3?-adenosine overhangs by incubation with Taq DNA polymerase (Gibco) and dATP. These molecules were then cloned into the pGem-T Easy vector (Promega). Sequencing of the clones confirmed the trimolecular nature of the original ligation products (data not shown).

Assay calibration with synthetic inserts containing increasing numbers of A5 tracts

DNA inserts used for calibration are also displayed in Figure 2 (duplexes 2?8). This set of inserts was chosen to provide a range of intrinsic curvatures caused by the interaction of one or more A5 tracts. The magnitude and direction of DNA curvature in A5 tracts has been well characterized (reviewed in 9). Duplex insert 1 contains no A5 tracts and is expected to be linear. Duplex inserts 2?4 each contain one A5 tract, separated by different distances from the 3?-end of the duplex. Duplex inserts 5?7 each contain two phased A5 tracts separated by different distances from the 3?-end of the duplex. Duplex insert 8 contains three phased A5 tracts. Electrophoresis of duplex inserts 1?8 ligated between L and each of the five right arms R-A to R-E therefore allows exploration of the dependence of electrophoretic mobility on the magnitude and position of intrinsically curved loci in relation to the position of the three phased A5 tracts in R-A to R-E.

Electrophoretic phasing analyses were employed to measure changes in mobility due to ligation of the synthetic inserts between L and each of the five right arms R-A to R-E. As the helical phasing between the two elements (reference A5 tracts in R-A to R-E versus test A5 tracts in the synthetic insert) changes, electrophoretic mobility is altered. When curvature loci are aligned in cis gel mobility is minimized. The extent to which gel mobility is reduced in each probe is interpreted as a measure of the magnitude and direction of DNA curvature contributed by the DNA duplex insert.

Representative data from calibration of the phasing assay with inserts containing differing numbers of phased A5 tracts are shown in Figure 3. Duplex inserts 1, 4, 7 and 8 (Fig. 2) were ligated between L and each of the five right arms R-A to R-E and electrophoresed through an 8% native polyacrylamide gel as described in Materials and Methods (Fig. 3A). Electrophoresis of products containing insert 1 (Fig. 3A, lanes 1?5) result in subtle mobility differences, suggesting little DNA curvature in duplex insert 1.

A different phasing profile is seen for duplex insert 4 (Fig.?3A, lanes 6?10). The mobilities of these products displayed an obvious dependence on the position of the A5 tract in relation to the A5 tracts of R-A to R-E. Mobility retardation is maximal for products containing R-B (Fig. 3A, lane 7), indicating that the loci of curvature are most nearly in phase. Mobility retardation is minimized in R-D and R-E (Fig.?3A, lanes 9 and 10), where the loci of curvature are out of phase.

Electrophoresis of products derived from duplex insert 7 (Fig. 3A, lanes 11?15) and duplex insert 8 (Fig. 3A, lanes 16?20) display greater phase-dependent mobilities. As the number of A5 tracts in the synthetic duplex insert is increased, mobility anomalies are enhanced, as expected. As with insert 4, the most retarded product was R-B and the least retarded products were R-D and R-E.

The gel mobility of each probe was analyzed as described previously (18). Relative mobility, ?rel (equation 1), was plotted against the spacing (in bp) between the double-stranded region of the duplex insert and the center of the 5? A5 tract in each of the five right arms R-A to R-E. The results are depicted in Figure 3B. Using techniques derived by Kerppola and co-workers (18), the relative differences in probe mobilities were transformed (equation 2) into estimates of DNA curvature magnitudes (Fig. 4). Analysis of the electrophoretic mobilities of probes containing duplex inserts 1, 4, 7 and 8 reveals a linear relationship between the measured phasing amplitude and the predicted degree of DNA curvature over this range:

APH = 0.026 + 0.0047CP, r2 = 0.991 3

where APH is the amplitude of the phasing function and CP is the predicted curvature (deflection of the helix axis in degrees) of the DNA duplex insert.

In addition to calibrating the relationship between measured phasing amplitude and A5 tract curvature, we also investigated the effect of changing the distance between A5 tracts in the DNA duplex inserts and the reference A5 tracts in each of the five right arms R-A to R-E on the measured gel mobility anomalies. We measured the resultant phasing amplitudes following ligation of duplex inserts containing a single A5 tract distal (2), medial (3) or proximal (4) with respect to the reference A5 tracts in each of the five right arms R-A to R-E (Fig. 2).

The results of this analysis are also shown in Figure 4. The measured phasing amplitudes confirmed our expectations: the phasing amplitude was greatest for insert 4, where the A5 tract in the duplex insert is closest to the reference A5 tracts. Insert 3 yielded an intermediate value and insert 2, containing an A5 tract most distant from the reference A5 tracts, produced the smallest gel mobility anomaly upon electrophoresis.

We repeated these studies with duplex inserts that contained two phased A5 tracts located either distal and centered (5), distal and proximal (6) or centered and proximal (7) with respect to the reference A5 tracts in each of the five right arms R-A to R-E (Fig. 2). The measured phasing amplitudes confirmed our expectations: amplitudes were greatest for insert 7, where the A5 tracts in the insert are closest to the reference A5 tracts. Insert 6 was intermediate in character and insert 5, containing the most distant A5 tracts, produced the smallest gel mobility anomaly upon electrophoresis (Fig. 4).

Our results agree well with those obtained by Kerppola in a thorough study of the phase-dependent mobility variation of DNA fragments containing two intrinsic bends (19). This study demonstrated that when the separation between two bends was increased, the phase-dependent mobility anomaly of the fragments decreased. Kerppola also showed that for DNA fragments with long flanking sequences, two closely spaced loci of curvature that are in phase caused a mobility anomaly similar to that due to a single locus of curvature representing the sum of the two curvatures (19). However, when the separation between the loci of curvature was increased, their effect on electrophoretic mobility decreased. Thus, two loci of curvature that are in phase but separated by a long DNA spacer do not interact in electrophoretic phasing assays to the same extent as two closely juxtaposed bends (19).

Quantitatively, our analysis yields similar results. The sum of phasing amplitudes computed from pairs of duplex inserts 2 + 7, 3 + 6 and 4 + 5 are very similar (0.244, 0.228 and 0.257, respectively). This result indicates the additivity of two closely spaced loci of curvature in our system. The summed phasing amplitudes are slightly less than that determined for insert 8 (0.286), which contains three phased A5 tracts. This small difference may be attributed to the distance between the A5 tracts in the duplex insert and the reference A5 tracts.

We conclude that calibration of differential electrophoretic anomalies to predicted DNA curvatures allows the potential quantitative application of this semi-synthetic phasing assay to a wide variety of other studies. We present two examples below.

DNA bending by phosphate neutralization

A key advantage of the proposed semi-synthetic phasing approach is the ability to study the shape of a single synthetic DNA duplex containing chemical modifications. Because the phasing assay is constructed by ligating different sets of prefabricated, variable DNA arms to a single synthetic DNA duplex, the expense and effort associated with synthesis, purification and assembly of multiple modified duplexes is avoided. Our previous experiments (11,14,20?22) each typically required synthesis of 16?20 oligonucleotides to allow curvature estimates based on three helical phasings of a test sequence relative to a standard locus of curvature. We sought to determine if a single synthetic duplex could provide information based on five phasings using the semi-synthetic phasing strategy.

We therefore considered changes in DNA shape imparted by asymmetrical neutralization of the phosphate backbone due to partial methylphosphonate substitution. Methylphosphonate substitution has been shown to induce DNA curvature (11). We wanted to determine if these effects would be reflected in changes in ligation product mobility in our phasing assay.

We designed DNA duplex inserts 9 and 10 (Fig. 2) to correspond to previously characterized DNA duplexes containing neutral methylphosphonate substitutions positioned across one minor groove (insert 10), separated by 9.5 bp from the center of the A6 tract curvature (0.5 bp 3? of the center of the A6 tract) or its unmodified control sequence (insert 9, Fig. 2; 11). Insert 7 served as a reference and contained two phased A5 tracts (Fig.?2). Inserts 7, 9 and 10 were ligated between L and each of the five right arms R-A to R-E and products were electrophoresed through a native polyacrylamide gel. Figure 5A displays a representative profile of ligation products obtained and their corresponding mobilities. Electrophoresis of probes containing duplex insert 7 (Fig. 5A, lanes 1?5) results in the expected phase-dependent mobility differences due to the position of the A5 tracts, relative to the A5 tracts in R-A to R-E.

A different phasing profile is seen for duplex insert 9 (Fig.?5A, lanes 6?10). These products display reduced mobility anomalies. This suggests that the unmodified sequence contains little intrinsic curvature relative to that possessed by the A5 tract in insert 7. Mobility retardation is maximal for probes containing R-B (Fig. 5A, lane 7) and is minimized in R-D and R-E (Fig. 5A, lanes 9 and 10), as observed for insert 7.

Electrophoresis of products derived from neutralized duplex insert 10 (Fig. 5A, lanes 11?15) demonstrated pronounced differences among mobilities of the probes containing unmodified insert 9 (Fig. 5A, lanes 6?10). Mobility differences approach those observed for insert 7, confirming that methylphosphonate substitution at the indicated positions of insert 10 (Fig. 2) induces substantial DNA curvature.

Transformation of methylphosphonate phasing data (see for example Fig. 5B) for inserts 9 and 10 and fitting to equation 3 yields a curvature estimate of 12.9 ? 1.7? as a result of methylphosphonate substitution. This value agrees fairly well with our previous estimate of 17.8? (10,11). The semi-synthetic phasing assay also detects the direction of the bend resulting from methylphosphonate substitution. This information is revealed by the spacing at which the fitted cosine function is at a minimum (Fig. 5B). We deduced from insert 9 the base pair spacing that defines the cis orientation between the center of the A6 tract in 9 and the center of the 5? A5 tract in the array of the five right arms R-A to R-E. This distance is calculated as a sum. First, we note the base pair spacing between the 3?-terminus of the double-stranded region of duplex insert 9 and the center of the 5? A5 tract in R-A to R-E at which the cosine function is at a minimum (Fig. 5B). Second, we count base pairs in a 5? direction to the center of curvature of the A6 tract in 9. This total spacing was found to be ?32.8 bp. Because 32.8 bp must therefore represent an integral number of DNA helical turns, the average DNA helical repeat over this region is estimated at ?10.9 bp per turn. If one then examines insert 10, two turns of the helix from the center of curvature of the A5 tract (0.5 bp 3? of the center of the A5 tract) in the array of the five right arms R-A to R-E yields a spacing of 21.8 bp. From Figure 5B the optimal cis spacing between the methylphosphonate patch and the A5 tract is observed to be 22.4 bp. This defines the true bend position as an additional ?0.6 bp 5? from the assigned center of the neutralized face. This value agrees well with our previous estimate of bending toward the minor groove in a reference frame 0.8 bp shifted 5? from the center of?the neutralized face (10). We therefore conclude that the semi-synthetic phasing assay yields a reasonably accurate measure of both the magnitude and direction of curvature induced by methylphosphonate substitution.

DNA bending by GCN4 charge variants

Traditional phasing assays of DNA bending by proteins require the cloning of protein binding sites into a series of phasing vectors, followed by sequencing, plasmid preparation, restriction digests, radiolabeling, probe purification, protein binding and electrophoresis. We considered the possibility that a simple set of prefabricated left and right DNA phasing arms would facilitate assembly of phasing probes. Thus, a single radiolabeled synthetic DNA duplex bearing the protein binding site of interest is prepared and ligated between the proper combinations of arms. The resulting probes are incubated with protein and studied by gel mobility shift assays. In the present study we have assayed protein binding without further probe purification.

Apparent DNA bending by variants of the DNA-binding domain of recombinant GCN4, a basic leucine zipper transcription factor, has been studied previously by our laboratory (23; P.R.Hardwidge and L.J.Maher, unpublished results). We previously hypothesized that if interphosphate repulsions are important in determining DNA shape, substitution of three cationic lysine residues (KKK) or three anionic glutamate residues (EEE) for the three neutral amino acids (PAA) of GCN4 might result in bending of the DNA to which GCN4 binds. Amino acid substitutions were made at positions ?26 to ?24 of a peptide (71 amino acids) representing the DNA-binding domain of GCN4, where the first leucine of the zipper heptad repeat is defined as residue +1 (24). Electrophoretic phasing experiments supported this hypothesis: cationic amino acid substitutions resulted in an apparent DNA bend toward the protein-bound face, while anionic amino acid substitutions caused apparent DNA bending away from the protein-bound face (23; P.R.Hardwidge and L.J.Maher, unpublished results).

We therefore explored DNA bending induced by GCN4 peptide charge variants (23,25; P.R.Hardwidge and L.J.Maher, unpublished results). Insert 11 (Fig. 2), containing the GCN4 binding site (AP-1), was ligated between L and each of the five right arms R-A to R-E and the resulting products were resolved through a native polyacrylamide gel. Representative phasing data are presented in Figure 6A. The faster migrating species in Figure 6A are due to protein binding to partial ligation products. Lanes 1?5 reveal the mobilities of probes containing the AP-1 duplex (insert 11) in the absence of protein. Lanes 6?10 display the mobilities of complexes involving the binding of PAA to the phasing probes. Subtle mobility differences are observed among complexes. In complexes containing EEE (Fig. 6A, lanes 11?15) mobility differences among probes are greater than with PAA. Probes containing R-A and R-B migrated significantly faster when complexed with EEE than with PAA. A very different profile of probe mobilities was seen in complexes with KKK (Fig. 6A, lanes 16?20). In these complexes the general profile of mobility differences was shifted by one half helical turn of DNA, consistent with DNA bending by KKK in a direction opposite to that induced by EEE.

Electrophoretic mobilities were normalized as described above and plotted against the distance in base pairs between the center of the AP-1 site and the center of curvature in the phased A5 tracts in R-A to R-E. The results are depicted in Figure 6B. Transformation of GCN4 phasing data into bending estimates as described above (using equation 3) yields values of 2.5 ? 1.8? (PAA), 12.6 ? 2.2? (EEE) and 13.7 ? 1.0? (KKK). The intrinsic curvature of insert 11 was estimated to be 10.1 ? 0.7?. Previous estimates of bend angles induced by peptide binding in an independent electrophoretic phasing assay were 2? (PAA), 10? (EEE) and 23? (KKK) (P.R.Hardwidge and L.J.Maher, unpublished results). The intrinsic curvature of the AP-1 site in insert 11 was previously estimated to be 4?6? (12; P.R.Hardwidge and L.J.Maher, unpublished results).

Figure 6B also allows analysis of the direction of DNA bending induced upon GCN4 binding. The minima of the fitted cosine functions for unbound probe 11 and for 11 when bound by PAA and EEE are all comparable. This suggests that bending by PAA and EEE is in the same direction as in the free AP-1 site, as has been proposed previously (23). A minimum shifted by ?5 bp was observed in complexes with KKK, suggesting bending of the DNA in a nearly opposite direction. Thus, our estimates of GCN4 charge variant-induced DNA bending determined in the semi-synthetic phasing assay correspond reasonably well to those obtained in an independent electrophoretic assay.

Summary

Five aspects of our semi-synthetic phasing probe design deserve particular emphasis. (i) Probe construction requires only one synthetic duplex and six PCRs with subsequent restriction endonuclease digestions, whereas the ligation ladder method requires the design of ?20 new oligonucleotides for each experiment. (ii) Five phasings are studied, compared to three in the conventional system. (iii) Phasing profiles can be calibrated to extract bend angles through the ligation of duplexes containing standards of intrinsic curvature. (iv) It is critical in conventional ligation ladder assays of DNA curvature that the exact helical repeat of the test sequence is established since proper phasing of test sequences must be maintained throughout the polymers. In contrast, the precise helical repeat of the synthetic duplex insert is of less consequence in the semi-synthetic probe design, as the sequence occurs only once. It is the helical repeat of the unmodified spacer DNA that is of great importance, and this parameter is invariant and can be established in advance. (v) Synthetic inserts can be relatively large.

In practice, the need for subcloning a protein binding site prior to new phasing studies is eliminated in our system. One needs only to synthesize an appropriate DNA duplex of interest and ligate the duplex between the left arm L and each of the five right arms R-A to R-E. These DNA arms are easily generated by BbsI digestion of the six parent plasmids. One might also construct the three DNA duplexes 4, 7 and 8 for calibration of equation 3 for each new electrophoretic system.

A number of limitations are also inherent in the design of our semi-synthetic phasing assay. The resultant phasing amplitudes are to some extent dependent on the position of a locus of curvature in the probes, relative to the position of the reference A5 tracts in the five right arms R-A to R-E. This effect is shown in Figure 4 and has been discussed previously (19). The reduction in phasing amplitude as the distance between loci of curvature is increased has important implications in the design and analysis of our system. One must decide the position within a synthetic DNA duplex at which to place the locus of curvature to be studied. For a given phasing amplitude, placement of the locus nearest to the reference A5 tracts establishes an upper bound and placement far from the reference A5 tracts establishes a lower bound for predicted curvature in degrees. Therefore, for maximum sensitivity one must position the sequence to be studied near to the right arm. This also complicates the study of longer sequences. For a sequence of interest greater in length than a helical turn of DNA a diminished phasing amplitude is contributed by elements of curvature in the distal portion of the studied sequence. Thus, for a very long DNA duplex insert it may be possible to evaluate only the total intrinsic curvature present in the insert; resolution of large curvatures to individual base pairs may not be possible. However, the construction of several variants of a given DNA duplex insert might eliminate this problem.

As in ligation ladder assays (reviewed in 9), the presence of nicks in ligation products may alter gel mobilities in an unpredictable manner. The influence of nicks on gel mobility should be reduced in the semi-synthetic phasing assay, relative to the ligation ladder assay, because the semi-synthetic assay involves only two ligation steps, whereas ligation ladder products of interest typically involve ligation of six to nine DNA duplexes.

We note that despite its efficiency and convenience, the calibration of phasing amplitudes to DNA curvature may be less accurate than calibrations in the ligation ladder assay, primarily due to the effect of distance on phasing amplitude as shown in Figure 4. This issue remains to be explored fully.


CONCLUSION

We have developed a semi-synthetic phasing assay that offers several advantages relative to established comparative electrophoretic methods. This assay requires a reduced number of synthetic oligonucleotides, involves no cloning manipulations, is economical and can be applied to a wide variety of DNA curvature studies. Gel mobility retardation of phasing probes is empirically correlated with the predicted DNA curvature provided by A5 tracts. We also apply this system to previously studied examples of DNA curvature induced by DNA methylphosphonate substitution and apparent DNA bending induced by GCN4 charge variants. We propose this method as a versatile and rapid means of studying DNA curvature in other systems.



ACKNOWLEDGEMENTS

We thank A. Schepartz and D. Paollela for plasmid pDP-AP-1-21. We acknowledge M. Doerge of the Mayo Foundation Molecular Biology Core Facility for providing excellent oligonucleotide synthesis services. This work was supported by the Mayo Foundation and NIH grant GM54411 to L.J.M.


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Figures

[Figure ID: gnd102f1]
Figure 1 

Phasing assay design. Phasing probes are trimolecular ligation products (top) containing a synthetic DNA duplex insert (I) flanked by restriction fragments (L and R). Cohesive termini are indicated in bold. In the present studies each strand of the synthetic insert was 36 bases in length. DNA sequences of the left arm (L), synthetic DNA duplex insert (I) and right arms (R-A to R-E) are shown. Phased A5 tracts in the right arms are in black boxes with white lettering. Right arms differ only in the initial distance to the first A5 tract of the array. Top strand sequences are shown 5??3? (left to right).



[Figure ID: gnd102f2]
Figure 2 

DNA duplex inserts (I) under investigation. A5/6 tracts are in black boxes with white lettering. Neutral methylphosphonate internucleoside linkages are shown by black dots. The AP-1 site in duplex insert 11 is shown with a solid bar. Top strand sequences are shown 5??3? (left to right). The indicated overhanging termini allow selective ligation of I between L and each of the right arms R-A to R-E.



[Figure ID: gnd102f3]
Figure 3 

Calibration of the phasing assay with A5 tract duplex inserts. (A) Image obtained after native polyacrylamide gel electrophoresis of phasing probes containing duplexes 1, 4, 7 and 8. (B) Quantitative analysis of electrophoretic data for inserts 1 (filled circles), 4 (filled squares), 7 (open circles) and 8 (open squares). The relative mobility of each of the five phasing probes in a given protein complex (?rel) is plotted as a function of the spacing (in bp) between the double-stranded region of the duplex insert and the center of the 5? A5 tract in R-A to R-E and fitted to equation 2 as described in Materials and Methods.



[Figure ID: gnd102f4]
Figure 4 

Relationship between electrophoretic phasing amplitude and predicted DNA curvature. Phasing amplitudes (? SD) derived from curve fitting of gel mobility data to equation 2 are plotted against the predicted degrees of curvature in the synthetic insert (based upon a conventional estimate of 18? per A5 tract). DNA duplex insert number is indicated to the right of each data point. Filled circles correspond to probes in which A5 tracts are proximal. A fit of these data points to equation 3 is shown.



[Figure ID: gnd102f5]
Figure 5 

Detection of DNA curvature induced by phosphate charge neutralization. (A) Image obtained after native polyacrylamide gel electrophoresis of phasing probes containing duplex inserts 7, 9 and 10. (B) Quantitative analysis of electrophoretic data for duplex inserts 7 (open squares), 9 (filled squares) and 10 (open circles). A representative data set is shown. Typical standard deviations for phasing amplitudes were ?0.01, n = 3?6.



[Figure ID: gnd102f6]
Figure 6 

Detection of DNA bending induced by binding of GCN4 charge variants. (A) Image obtained after native polyacrylamide gel electrophoresis of phasing probes containing duplex insert 11, free or bound to GCN4 peptides PAA, EEE and KKK. (B) Quantitative analysis of electrophoretic data for duplex insert 11, free (filled circles) or bound to GCN4 peptides PAA (open circles), EEE (filled squares) and KKK (open squares). A representative data set is shown. Typical standard deviations for phasing amplitudes were ?0.01, n = 3?6.



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