Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.     

Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein · nucleic acid complexes

We used a mutant gene 5 protein (g5p) to assign and interpret overlapping CD bands of protein · nucleic acid complexes. The analysis of overlapping protein and nucleic acid CD bands is a common challenge for CD spectroscopists, since both components of the complex may change upon binding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mutant gene 5 protein that has an insignificant change in tyrosine optical activity at 229 nm upon binding to nucleic acids. We have studied the interactions of the mutant Y34F g5p (Tyr-34 substituted with phenylalanine) with poly[r(A)], poly[d(A)], and fd single-stranded DNA (ssDNA). Our results showed the following: (1) The 205–300 nm spectrum of poly[r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentially the sum of the spectra of poly[r(A)] at a high temperature plus the spectrum of the free protein, except for a minor negative band at 257 nm. (2) The spectra of poly[d(A)] and fd ssDNA saturated with the mutant protein at a P/N = 0.25, minus the spectra of the free nucleic acids at a high temperature, also essentially equaled the spectrum of the free protein in the 205–245 nm region. (3) While the overall secondary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of individual aromatic residues. (4) Nucleic acids complexed with the g5p are unstacked (as if heated) and (in the cases of the DNAs) perturbed as if part of a dehydrated double-stranded DNA. (5) Difference spectra revealed regions of the spectrum specific for the particular nucleic acid, the protein, and whether g5p was bound to DNA or RNA. © 1997 John Wiley and Sons, Inc. Biopoly 42: 337–348, 1997     

Specificity of the binding of fd gene 5 protein to polydeoxyribonucleotides

The long-wavelength circular dichroism (CD) changes induced by binding of fd gene 5 protein to the alternating DNA sequences poly[d(A-C)] and poly[d(C-T)] were similar to those induced by the protein complexed with the homopolymers poly[d(A)], poly[d(C)], and poly[d(T)]. The fd gene 5 protein showed different binding affinities for the various polymers. The affinity for the alternating sequences was not compositionally weighted with respect to the affinities for the homopolymers, indicating that both base composition and base sequence of the template are important for the binding of fd gene 5 protein.     

Specificity of the Binding of fd Gene 5 Protein to Polydeoxyribonucleotides

Abstract

The long-wavelength circular dichroism (CD) changes induced by binding of fd gene 5 protein to the alternating DNA sequences poly[d(A-C)] and poly [d(C-T)] were similar to those induced by the protein complexed with the homopolymers poly[d(A)], poly[d(C)], and poly[d(T)]. The fd gene 5 protein showed different binding affinities for the various polymers. The affinity for the alternating sequences was not compositionally weighted with respect to the affinities for the homopolymers, indicating that both base composition and base sequence of the template are important for the binding of fd gene 5 protein.     

The binding affinity of Ff gene 5 protein depends on the nearest-neighbor composition of the ssDNA substrate

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.     

Effects of ssDNA sequences on non-sequence-specific protein binding

The circular dichroism (CD) spectra of single-stranded DNAs (ssDNAs) are significantly perturbed by the binding of single-stranded DNA binding proteins such as the Ff bacteriophage gene 5 protein (g5p) and the A domain of the 70 kDa subunit of human replication protein A (RPA70-A). These two proteins have similar OB-fold secondary structures, although their CD spectra at wavelengths below 250 nm differ greatly. The spectrum of g5p is dominated by a tyrosyl L(a) band at 229 nm, while that of RPA70-A is dominated by its beta secondary structure. Despite differences in their inherent spectral properties, these two proteins similarly perturb the spectra of bound nucleic acid oligomers. CD spectra of free, non-protein-bound ssDNAs are dependent on interactions of the nearest-neighboring nucleotides in the sequence. The CD spectra (per mol of nucleotide) of simple repetitive sequences 48 nucleotides in length and containing simple combinations of A and C are related by nearest-neighbor equations. For example, 3 x Deltaepsilon[d(AAC)(16)] = 3 x Deltaepsilon[d(ACC)(16)] + Deltaepsilon[d(A)(48)] - Deltaepsilon[d(C)(48)]. Moreover, nearest-neighbor equations relate the spectra of ssDNAs when they are bound by g5p, indicating that each type of perturbed nearest neighbor has a similar average structure within the binding site of the protein.     

Interaction of a dimeric distamycin analog with poly(dA)poly(dT), poly[d(A–T)]poly[d(A–T)], and duplex O<Subscript>23</Subscript> at the origin of replication of the herpes simplex virus

The binding of a dimeric distamycin analog (Pt–bis–Dst) to poly[d(A–T)]poly[d(A–T)], poly(dA)poly(dT), and duplex O₂₃ with the sequence 5’-GCCAATATATATATATTATTAGG-3’, which occurs at the origin of replication (OriS) of the herpes simplex virus, was studied via UV and CD spectroscopy. The synthetic polyamide differs from the natural antibiotic in having two distamycin moieties that are linked via a glycine cis-diamino platinum group. The Pt–bis–Dst binding to poly[d(A–T)]poly[d(A–T)] and poly(dA)poly(dT) reached saturation at approximately one ligand molecule per eight bp. As the ligand–base pair ratio further increased, the maximum wavelength band tended to shift toward longer wavelengths in the CD spectra of complexes with poly[d(A–T)]poly[d(A–T)] and a shoulder appeared in the 290–310 nm spectral region that was absent from the CD spectra of complexes with lower ligand coverages. At higher ligand–oligonucleotide molar ratios, Pt–bis–Dst could bind to poly[d(A–T)]poly[d(A–T)] in the form of hairpins or associations that result from interactions between the distamycin moieties of two neighbor Pt–bis–Dst molecules. The structures of the complexes were stabilized by interactions between the pirrolcarboxamide moieties of two Pt–bis–Dst molecules absorbed on adjacent overlapping binding sites. The interactions could also be responsible for the concentration-dependent spectral changes that were observed during the formation of a complex between Pt–bis–Dst and poly[d(A–T)]poly[d(A–T)]. Spectral changes were almost absent in the case of Pt–bis–Dst binding to poly(dA)poly(dT). The binding of Pt–bis–Dst to duplex O₂₃ reached saturation at two ligand molecules per duplex, which contained a cluster of 18 AT pairs. At higher molar-concentration ratios, duplex CD spectra underwent changes similar to those that were observed for Pt–bis–Dst binding to poly[d(A–T)]poly[d(A–T)]. Testing Pt–bis–Dst for antiviral activity identified 1.5 μg/mL as a concentration that halved the cytopathic effect of the herpes simplex virus on Vero E6 cells; the selectivity index of antiviral action was 65; cytotoxicity was relatively low. The Pt–bis–Dst concentration that caused the death of approximately half of the cells was estimated at 100 μg/mL.     

Identification of Tyr(290(6.58)) of the human gonadotropin-releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: importance for high-affinity binding and receptor activation

Molecular modeling showed interactions of Tyr (290(6.58)) in transmembrane domain 6 of the GnRH receptor with Tyr (5) of GnRH I, and His (5) of GnRH II. The wild-type receptor exhibited high affinity for [Phe (5)]GnRH I and [Tyr (5)]GnRH II, but 127- and 177-fold decreased affinity for [Ala (5)]GnRH I and [Ala (5)]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe (5)]GnRH I, GnRH II and [Tyr (5)]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr (290(6.58)) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr (290(6.58)) to Gln increased affinity for Tyr (5)-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr (5) of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr (290(6.58)) side chain with Tyr (5) of GnRH I and His (5) of GnRH II. Tyr (290(6.58)) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.     

Individual tyrosine side-chain contributions to circular dichroism of ribonuclease

Experimental and theoretical studies using site-directed mutants of ribonuclease A (RNase A) offer more extensive information on the tyrosine side-chain contributions to the circular dichroism (CD) of the enzyme. Bovine pancreatic RNase A has three exposed tyrosine residues (Tyr73, Tyr76, and Tyr115) and three buried tyrosine residues (Tyr25, Tyr92 and Tyr97). The difference CD spectra between the wild type and the mutants at pH 7.0 (Deltaepsilon(277,wt) - Deltaepsilon(277,mut)) show bands with more negative DeltaDeltaepsilon(277) values for Y73F and Y115F than those for Y25F and Y92F and bands with positive DeltaDeltaepsilon(277) values for Y76F and Y97F. The theoretical calculations are in good semiquantitative agreement for all the mutants. The pH difference spectrum (pH 11.3-7.0) for the wild type shows a negative band at 295 nm and an enhanced positive band at 245 nm. The three mutants at buried tyrosine sites and one mutant at an exposed tyrosine site (Y76F) exhibit pH-difference spectra that are similar to that of the wild type. In contrast, two mutants at exposed tyrosine sites (Y73F and Y115F) exhibit diminished 295-nm negative bands and, instead of positive bands at 245 nm, negative bands are observed. Our results indicate that Tyr73 and Tyr115, two of the exposed tyrosine residues, are the largest contributors to the 277- and 245-nm CD bands of RNaseA, but the buried tyrosine residues and the one remaining exposed residue also contribute to these bands. Disulfide contributions to the 277- and 240-nm bands and the peptide contribution to the 240-nm band are confirmed theoretically.     

The binding of fd gene 5 protein to polydeoxynucleotides: evidence from CD measurements for two binding modes

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly[d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)20, d(pA)7, and d(pT)7). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometries depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 +/- 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)] (with the first endpoints giving n = 4.1 4.0, 4.0, and 4.1 +/- 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non-interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol. Struct. and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex. We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A-and/or T-containing DNAs. Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20 degrees C, appeared to interact with one another in much the same manner as in the free polymers at 50 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyr61的芳香族侧链对稳定瘦素的结构至关重要

为了研究第61位Tyr（Tyr61）在瘦素（leptin）结构与功能中的作用,构建了2个瘦素突变体Y61F与Y61Q并对其进行了功能分析．Y61F突变体瘦素显示出与野生型瘦素相同的天然凝胶电泳迁移率及相似的折叠效率,而Y61Q突变体瘦素则显示出明显慢的电泳迁移率且较野生型瘦素更难于折叠．受体结合及免疫活性测定显示, Y61F突变体保留了野生型瘦素的大部分生物学活性,而Y61Q突变体仅保留了野生型瘦素16％的受体结合活性及30％的免疫活性．圆二色性分析及二级结构估算表明,Y61F突变体具有与野生型瘦素几乎一样的二级结构组成,而Y61Q突变体的结构则较野生型瘦素更为松散.本研究表明,Tyr61的芳香族侧链被包埋于分子内部的疏水区域中对稳定瘦素结构及其发挥生理功能至关重要,而Tyr61上的羟基在这一过程中并不起重要作用．     

Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.     

Circular dichroic properties of the tyrosine residues in tetrazole analogues of opioid peptides.

CD studies on tetrazole analogues of opioid peptides show that peptides sharing the same N-terminal sequence, H-TyrPsi[CN(4)]Gly-, give very large Cotton effects of the Tyr side chain in the near-UV region. CD spectra of five such peptides: H-TyrPsi[CN(4)]Gly-Gly-Phe-Leu-OH (I), H-TyrPsi[CN(4)]Gly-Phe-Pro-Gly-Pro-Ile-NH(2) (II), H-TyrPsi[CN(4)]Gly-Phe-Pro-NH(2) (III), H-TyrPsi[CN(4)]Gly-Phe-Gly-Tyr-Pro-Ser-NH(2) (IV), and H-TyrPsi[CN(4)]Gly-Phe-Asp-Val-Val-Gly-NH(2) (V), and two others for comparison: H-Tyr-GlyPsi[CN(4)]Gly-Phe-Leu-OH (VI) and H-TyrPsi[CN(4)]Ala-Phe-Gly-Tyr-Pro-Ser-NH(2) (VII), were measured in methanol, 2,2,2-trifluoroethanol, and water at different pH values. The spectra show that the conformations of the Tyr(1) residue in peptides I-V are very similar in all solvents used but differ distinctly from those observed for VI and VII. Strong Tyr bands in the aromatic region result probably from the rigid structure of the common N-terminal part of peptides I-V. These bands are weaker for IV, which maybe due to the presence of a second Tyr residue in that peptide, giving an opposite contribution to the CD spectrum as that arising from Tyr1. It seems that the rigid structure of the N-terminal part of I-V results from the interaction of the Tyr(1) side chain and the tetrazole ring. The CD bands of the Tyr residues of VI and VII are much smaller than those of I-V in all solvents, except VII in trifluoroethanol (TFE) where Tyr bands comparable in intensity to those of I-V are observed. This spectral property may derive from the same sign contribution of both Tyr residues of VII to the CD spectrum.     

Interaction of the HMG1 protein with nucleic acids

Binding constants have been measured for the interaction of the protein HMG1 with native DNA, denatured DNA and a number of polynucleotides at near-physiological ionic strengths, using gel filtration and thermal denaturation. The interaction of HMG1 with DNA is shown to be noncooperative and reversible. Nucleic acids form the following series in order of increasing binding constants: poly(U) integral of poly(A) less than poly(dA) less than dsDNA integral of poly(dA) X poly(dT) integral of poly(dG) X poly(dC) much less than poly[d(A-T]) integral of ssDNA.     

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Abstract

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly(d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)₂₀, d(pA)₇, and d(pT)₇). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometrics depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 ± 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)) (with the first endpoints giving n = 4.1, 4.0, 4.0, and 4.1 ± 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non- interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol Struct, and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex.

We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A- and/or T-containing DNAs, Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20°C, appeared to interact with one another in much the same manner as in the free polymers at 50°C. Finally, we found that the protein tyrosyl CD band at 228.5 nm decreased 39-42% when the protein bound to poly[d(A)] or poly[d(T)], but this band decreased no more than 9% when the gene 5 protein bound to short A- or T-containing oligomers. Thus, at least one tyrosyl residue has a significantly altered optical activity only when the DNA substrate is long enough either to cause a transition to a different protein conformation or to allow additional protein-protein contacts between adjacent helical turns of the DNA-protein complex.     

Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences

The relative immunogenicities of the poly[d(G-C)] and poly[d(A-C) · d(G-T)] families of helices have been determined. The specificities of the resultant immunoglobulins have been characterized for recognition of different synthetic and natural left-handed sequences and conformations. Certain modifications of poly[d(G-C)] in the sugar-phosphate bacbone and cytosine C-5 potentiate the right(R)-to-left(L) (B → Z) transition under physiological conditions. The resulting polynucleotides, poly[d(G^S-C)], poly[d(G-io⁵C)], poly[d(G-br⁵C)] and poly[d(G-m⁵C)], are also highly immunogenic. In contrast, DNAs incapable of assuming the left-handed conformation under physiological salt concentrations are weakly or non-immunogenic. These include unmodified poly[d(G-C)] as well as members of the poly[d(A-C) · d(G-T)] family of sequences bearing pyrimidine C-5 substitutions (methyl, bromo, iodo). These polynucleotides undergo the R → L isomerization under more stringent ionic and thermal conditions.The specificities of purified polyclonal and monoclonal anti-Z DNA immunoglobulins (IgG) were measured by binding to radiolabeled polynucleotides, by electrophoretic analysis of IgG bound to covalent closed circular DNAs, and by immunofluorescent staining of polytene chromosomes. The salt-induced left-handed forms of poly[d(G-C)] and its derivatives (including the cytidine C-5 methyl, bromo, iodo, and N-5 aza substituted polynucleotides) and of the modified poly[d(A-C) · d(G-T)] polymers are bound to varying degrees by different antibodies. The patterns of substrate recognition demonstrate the existence of several antigenic domains in left-handed DNAs, including the helix convex surface and the sugar-phosphate backbone. Substitutions in these regions can produce enhancing (required substitutions), neutral, or inhibitory effects on subsequent IgG binding. Additionally, certain modifications of either the convex surface of Z DNA at the C-5 position of cytidine (i.e. a methyl group) or of the backbone (i.e. phosphorothioate substitution) can lead to polymorphic lefthanded conformations that are compatible with antibody binding when present individually but not in combination. The recognition patterns exhibited with DNA substrates from the two DNA families indicate that some, but not all, IgGs show specificity for different nucleotide sequences.The anti-Z DNA IgGs were used to probe for specific left-handed Z DNA determinants on plasmid (e.g. pBR322) or viral (e.g. simian virus 40 (SV40)) DNAs and on the acid-fixed polytene chromosomes of dipteran larvae. At their extracted superhelical density, the negatively supercoiled form I, but not the relaxed, nicked, or linear forms of all tested plasmid and viral DNAs specifically bind sequence-independent anti-Z IgGs. Dimers, trimers and higher oligomers of form I DNA cross-linked by bivalent anti-Z IgGs are formed with numerous (e.g. φX174, SV40, pBR322) genomes. Their occurrence depends upon IgG concentration and specificity, the conditions of ionic strength and temperatures and the DNA genome. The IgG cross-linked DNA multimers are converted to monomers by dithiothreitol reduction. Sequence-independent monovalent anti-Z Fab fragments bind form I DNA but do not generate oligomeric species. Multimers of order >2 indicate the existence of at least two anti-Z Ig binding sites per molecule, as in the case of SV40. IgGs differ in their ability to form stable complexes with some sites on natural DNAs, presumably due to their sequence and conformation binding specificities. A differential binding of these antibodies is also observed in certain bands of polytene chromosomes, such as the telomeric regions that are involved in chromosome associations.     

Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA

The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.     

Effect of cesium on the volume of the helix-coil transition of dA.dT polymers and their ligand complexes

The pressure dependence of the helix–coil transition of poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of NaCl and CsCl at concentrations between 10 and 200 mM is reported and used to calculate the accompanying volume change. We also investigated the binding parameters and volume change of ethidium bromide binding with poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of these two salts. The volume change of helix–coil transition of poly(dA)∙poly(dT) in Cs⁺-containing solutions differs by less than 1 cm³ mol^− 1 from the value measured when Na⁺ is the counter-ion. We propose that this insensitivity towards salt type arises if the counter-ions are essentially fully hydrated around DNA and the DNA conformation is not significantly altered by salt types. Circular dichroism spectroscopy showed that the previously observed large volumetric disparity for the helix–coil transition of poly[d(A-T)]·poly[d(A-T)] in solutions containing Na⁺ and Cs⁺ is likely result of a Cs⁺-induced conformation change that is specific for poly[d(A-T)]·poly[d(A-T)]. This cation-specific conformation difference is mostly absent for poly(dA)∙poly(dT) and EB bound poly[d(A-T)]·poly[d(A-T)].     

A Phage Single-Stranded DNA (ssDNA) Binding Protein Complements ssDNA Accumulation of a Geminivirus and Interferes with Viral Movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.