Oligodeoxyribonucleotide length and sequence effects on intermolecular purine-purine-pyrimidine triple-helix formation.

The binding of guanosine/thymidine-rich oligodeoxyribonucleotides containing various deletions, extensions, and point mutations to polypurine DNA targets was investigated by DNase I footprinting. Intermolecular purine-purine-pyrimidine triple-helical DNA formation was best achieved using oligonucleotides 12 nucleotides in length. Longer oligonucleotides were slightly weaker in binding affinity, whereas shorter oligonucleotides were considerably weaker. Oligonucleotide extensions had a slight effect on triplex formation, while single point mutations located near the oligonucleotide ends had a greater effect. In the cases of extensions and point mutations, changes to the 3' end of the oligonucleotide had a consistently greater effect on triplex formation than changes to the 5' end. Such differences in triplex-forming ability were not caused by an intrinsic property of these oligonucleotides, since the same point mutated oligonucleotides could bind with high affinity to duplex DNAs containing complementary sites. Taken together, our data suggest that there may be an asymmetry involved in the process of purine-motif triplex formation, with interactions between the 3' end of the oligonucleotide and complementary sequences on the target duplex DNA being dominant.     

Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin.

IgE-binding protein (epsilon BP) was originally identified by virtue of its affinity for IgE. It is now known to be a beta-galactoside-binding lectin with the characteristic of an S-type carbohydrate recognition domain. The protein is composed of two domains: the amino-terminal domain consisting of tandem repeats and the carboxyl-terminal domain containing sequences shared by other S-type carbohydrate recognition domains. The amino-terminal domain also contains a number of potential recognition sites for collagenase cleavage. In this study, human epsilon BP was first expressed in Escherichia coli, and the carboxyl-terminal domain (epsilon BP-C) was then generated by collagenase digestion of epsilon BP. By equilibrium dialysis, the association constants of epsilon BP and epsilon BP-C for lactose were found to be similar (6.0 +/- 0.70) x 10(4) M-1 and (4.7 +/- 0.27) x 10(4) M-1, respectively. Both polypeptides contain only one lactose-binding site/molecule. By an assay involving binding of 125I-labeled epsilon BP or epsilon BP-C to solid phase IgE, and inhibition of this binding by saccharides, it was determined that epsilon BP-C retains the saccharide specificity of epsilon BP. Importantly, although unlabeled epsilon BP-C inhibited the binding of the radiolabeled epsilon BP to IgE, unlabeled epsilon BP caused increased binding to IgE, suggesting self-association among epsilon BP molecules. Oligomeric structures resulting from self-association of epsilon BP were confirmed by chemical cross-linking studies. Furthermore, epsilon BP possesses hemagglutination activity on rabbit erythrocytes, whereas epsilon BP-C lacks such activity. Based on these results, we propose a structural model for multivalency of epsilon BP: dimerization or oligomerization of epsilon BP occurs through intermolecular interaction involving the amino-terminal domain.     

Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences.

The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.     

DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.     

Theoretical analysis of ''addressed'' chemical modification of DNA.

Chemical "addressed" modification of DNA involves treatment of single-stranded DNA with oligonucleotides complementary to certain target sequences in this DNA and bearing a groupings reactive towards DNA bases. The binding of oligonucleotides can occur both at completely (specific) and incompletely (nonspecific) complementary sites. We analyse the modification of a fragment that is flanked by two target sequences complementary to a given oligonucleotide address, contains no more such targets and has some randomly distributed sites for nonspecific binding. Conditions for the maximum ratio between specific and non-specific modification are determined. We find the probability of both target termini being specifically modified without any non-specific modification occurring within the fragment up to a given moment in time. Quantitative analysis is based on the use of known features of the specific and non-specific binding of an oligonucleotide to DNA sites. This analysis shows the possibility of specific cutting of DNA based on addressed modification.     

Generating highly labeled oligonucleotides for DNA-protein interaction

We developed a new strategy to prepare double-stranded oligonucleotides containing recognition sites for specific binding proteins to examine DNA-protein interactions in various assays (gel mobility shift, UV-crosslinking, and affinity chromatography). The advantages of our procedures are as follows. Only one strand needs to be synthesized using a commercial oligonucleotide synthesizer. The probes can be labeled to a high specific activity and the exact position of labeling can be chosen, which is necessary for UV-crosslinking studies. Furthermore, multimeric binding sites for efficient DNA affinity chromatography can easily be generated. It is also possible to precisely place modified bases without the need for chemical precursors. Using this protocol, more detailed information about the binding protein factors and their behavior in interaction with recognition sites can be obtained.     

Triple-helix formation is compatible with an adjacent DNA-protein complex.

The effect of oligonucleotide-directed triple-helix formation on the binding of a protein to an immediately adjacent sequence has been examined. A double-stranded oligonucleotide was designed with a target site for the binding of a pyrimidine oligonucleotide located immediately adjacent to the recognition sequence for the herpes simplex virus type 1 (HSV-1) origin of replication binding protein, which is encoded by the UL9 gene of HSV-1. Since the optimal conditions for the binding of the UL9 protein and the pyrimidine oligonucleotide to the duplex DNA are markedly different, a pyrimidine oligonucleotide was designed to optimize binding affinity and specificity for the target duplex oligonucleotide. Consideration was given to length and sequence composition in an effort to maximize triple-strand formation under conditions amenable to the formation of the UL9-DNA complex. Using gel mobility shift assays, a trimolecular complex composed of duplex DNA bound to both a third oligonucleotide strand and the UL9 protein was detected, indicating that the UL9-DNA complex is compatible with the presence of a triple helix in the immediately adjacent sequences.     

Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.

The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.     

Activation of restriction endonuclease EcoRII does not depend on the cleavage of stimulator DNA.

The restriction endonuclease EcoRII is unable to cleave DNA molecules when recognition sites are very far apart. The enzyme, however can be activated in the presence of DNA molecules with a high frequency of EcoRII sites or by oligonucleotides containing recognition sites: Addition of the activator molecules stimulates cleavage of the refractory substrate. We now show that endonucleolysis of the stimulator molecules is not a necessary prerequisite of enzyme activation. A total EcoRII digest of pBR322 DNA or oligonucleotide duplexes with simulated EcoRII ends (containing the 5' phosphate group), as well as oligonucleotide duplexes containing modified bases within the EcoRII site, making them resistant to cleavage, are all capable of enzyme activation. For activation EcoRII requires the interaction with at least two recognition sites. The two sites may be on the same DNA molecule, on different oligonucleotide duplexes, or on one DNA molecule and one oligonucleotide duplex. The efficiency of functional intramolecular cooperation decreases with increasing distance between the sites. Intermolecular site interaction is inversely related to the size of the stimulator oligonucleotide duplex. The data are in agreement with a model whereby EcoRII simultaneously interacts with two recognition sites in the active complex, but cleavage of the site serving as an allosteric activator is not necessary.     

DNA-binding specificity of the S8 homeodomain.

The murine S8 homeobox gene is expressed in a mesenchyme-specific pattern in embryos, as well as in mesodermal cell lines. The S8 homeodomain is overall similar to paired type homeodomains, but at position 50, which is crucial for specific DNA recognition, it contains a Gln, as is found in Antennapedia (Antp)-type homeodomains. We determined the DNA-binding specificity of the purified S8 homeodomain by in vitro selection of random oligonucleotides. The resulting 11-bp consensus binding site, ANC/TC/TAATTAA/GC resembles, but subtly differs from, the recognition sequences of Antp-type homeodomains. Equilibrium binding constants of down to 6.0 x 10(-10) M were found for binding of the S8 homeodomain to selected oligonucleotides. Using specific antibodies and an oligonucleotide containing an S8-site, we detected by band-shift two abundant DNA binding activities in mesodermal cell lines that correspond to S8 and two more that correspond to its close relative MHox. These S8 protein forms are differentially expressed in retinoic acid-treated P19 EC cells.     

Sequence preference in DNA binding: de novo designed helix–turn–helix metallopeptides recognize a family of DNA target sites

The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix–turn–helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K _d=12.6±1.9 μM) and Ca(II) (K _d=70±8 μM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5′-pur-T-pur-G-(G/C)-3′], and two target sites for CaP3W [5′-(A/T)-G-G-G-(T/C)-3′ and 5′-A-T-(G/T)-T-G-3′]. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein–DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.     

A damage-recognition protein which binds to DNA containing interstrand cross-links is absent or defective in Fanconi anemia, complementation group A, cells.

A DNA binding protein with specificity for DNA containing interstrand cross-links induced by 4,5',8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light has been identified in normal human chromatin. Protein binding to DNA was determined using a gel mobility shift assay and an oligonucleotide containing a hot spot for formation of psoralen interstrand cross-links. Specificity of the damage-recognition protein for cross-links was demonstrated both by a positive correlation between level of cross-link formation in DNA and extent of protein binding and by effective competition by treated but not undamaged DNA for the binding protein. Chromatin protein extracts from cells from individuals with the genetic disorder, Fanconi anemia, complementation group A (FA-A), which have decreased ability to repair damage produced by TMP plus UVA light, failed to show any protein binding to TMP plus UVA treated DNA. We have previously shown that these chromatin protein extracts contain a DNA endonuclease complex, pI 4.6, which specifically recognizes and incises DNA containing interstrand cross-links and which in FA-A cells is defective in its ability to incise this damaged DNA (Lambert et al. (1992) Mutation Res., 273, 57-71). Together, these findings suggest that the DNA binding protein identified is involved in recognition and repair of DNA interstrand cross-links.     

Affinity and sequence specificity of DNA binding and site selection for primer synthesis by Escherichia coli primase

Primase is an essential DNA replication enzyme in Escherichia coli and responsible for primer synthesis during lagging strand DNA replication. Although the interaction of primase with single-stranded DNA plays an important role in primer RNA and Okazaki fragment synthesis, the mechanism of DNA binding and site selection for primer synthesis remains unknown. We have analyzed the energetics of DNA binding and the mechanism of site selection for the initiation of primer RNA synthesis on the lagging strand of the replication fork. Quantitative analysis of DNA binding by primase was carried out using a number of oligonucleotide sequences: oligo(dT)(25) and a 30 bp oligonucleotide derived from bacteriophage G4 origin (G4ori-wt). Primase bound both sequences with moderate affinity (K(d) = 1.2-1.4 x 10(-)(7) M); however, binding was stronger for G4ori-wt. G4ori-wt contained a CTG trinucleotide, which is a preferred site for initiation of primer synthesis. Analysis of DNA binding isotherms derived from primase binding to the oligonucleotide sequences by fluorescence anisotropy indicated that primase bound to DNA as a dimer, and this finding was further substantiated by electrophoretic mobility shift assays (EMSAs) and UV cross-linking of the primase-DNA complex. Dissection of the energetics involved in the primase-DNA interaction revealed a higher affinity of primase for DNA sequences containing the CTG triplet. This sequence preference of primase may likely be responsible for the initiation of primer synthesis in the CTG triplet sites in the E. coli lagging strand as well as in the origin of replication of bacteriophage G4.     

Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.     

Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure.

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607. To identify and define the subdomains for these two functions, we created an extensive series of deletions and point mutations in an EBNA-1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to heterodimerize with a wild-type EBNA-1 (aa 459 to 641) Immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.     

NaeI endonuclease binding to pBR322 DNA induces looping.

Previous work has demonstrated the existence of both resistant and cleavable NaeI sites. Cleavable sites introduced on exogenous DNA can act in trans to increase the catalysis of NaeI endonuclease cleavage at resistant sites without affecting the apparent binding affinity of the enzyme for the resistant site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. This activation suggests allosteric regulation of NaeI cleavage by distant cis- and trans-acting sites in DNAs containing both resistant and cleavable sites. Plasmid pBR322 contains four NaeI sites, at least one of which is resistant to cleavage. Electron microscopy is used here to demonstrate that NaeI endonuclease simultaneously binds to multiple recognition sites in pBR322 DNA to form loops with NaeI protein bound at the loop's base. The maximum number of loops formed with a common base suggests four binding sites per enzyme molecule. Looping was inhibited by addition of enzyme-saturating amounts of double-stranded oligonucleotide containing an NaeI site, whereas another double-strand oligonucleotide without the NaeI site had no effect. The number of loops seen was not above background when double-stranded M13 DNA, which contains only a single NaeI recognition site, was used as substrate.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The triplex stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson-Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.