Triplet state properties of tryptophan residues in complexes of mutated Escherichia coli single-stranded DNA binding proteins with single-stranded polynucleotides.

Complexes of point-mutated E. coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues. Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions. (Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872). The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling. The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp. For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp. Trp54 displays a 2[E]transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak [D] - [E] signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue. Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated. Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues. This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine. The observed decrease in the Trp [D] values further confirms the stacking of the Trp residues with 5-BrU. Wave-length-selected ODMR experiments conducted on the [D[ + [E] transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites. Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed.     

Optically detected magnetic resonance of tryptophan residues in Escherichia coli ssb gene product and E. coli plasmid-encoded single-stranded DNA-binding proteins and their complexes with poly(deoxythymidylic) acid

Optically detected magnetic resonance (ODMR) spectroscopy has been applied to several single-stranded DNA-binding (SSB) proteins encoded by conjugative plasmids of enteric bacteria. Fluorimetric equilibrium binding isotherms confirm their preferential binding to single-stranded DNA and polynucleotides and reveal a limited protein solubility at low ionic strength. The plasmid SSB-like proteins show the highest affinity for polydeoxythymidylic acid; these complexes are the least sensitive to disruption by salt. ODMR data on these complexes suggest the existence of stacking interactions between tryptophan residue(s) and thymine bases, as evidenced by spectral red shifts of the tryptophan phosphorescence 0,0 band, reduction of the magnitude of D zero field splitting parameter, and a dramatic reversal of the polarity of the ODMR signals. Wavelength-selected ODMR results point to the existence of two distinct tryptophan sites in these complexes. The triplet state properties of the red-shifted site are drastically altered by its interaction with the thymine bases. The chromosomal Escherichia coli SSB protein-poly(dT) complex shows an additional tryptophan site with zero field splitting parameters similar to those of the free protein. This site can be attributed to Trp-135, which is missing in each of the other plasmid SSB proteins, suggesting that this particular residue is not involved in the interaction with polynucleotides.     

An IncY plasmid-encoded single-stranded DNA-binding protein from Escherichia coli shows the identical pattern of stacked tryptophan residues as the chromosomal ssb gene product

In an extension of earlier studies on the Escherichia coli plasmid-encoded single-stranded DNA-binding proteins pIP71a SSB, F SSB and R64 SSB [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) Biochemistry 26, 3347-3354; Casas-Finet, J. R., Khamis, M. I., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) J. Biol. Chem. 262, 8574-8593], we have investigated the binding of pIP231a SSB to natural and heavy-atom-derivatized single-stranded homopolynucleotides. Fluorimetric equilibrium binding isotherms indicate that pIP231a SSB has a greater solubility at low ionic strength than any other plasmid SSB protein investigated. Furthermore, its complex with mercurated poly(uridylic acid) [poly(Hg5U)] shows a greater resistance to disruption by salt than the other plasmid SSB complexes. Essentially complete binding of pIP231a SSB to poly(Hg5U) could be achieved, and time-resolved optically detected triplet-state magnetic resonance (ODMR) techniques could be applied to the complex. These methods allowed complete resolution of the three Trp chromophores of pIP231a SSB. Comparison of wavelength-selected ODMR results with those obtained for the poly(Hg5U) complex of a point-mutated chromosomal ssb gene product (Eco SSB) carrying substitutions of Phe for Trp [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Murphy, J. B. & Chase, J. W. (1987) J. Biol. Chem. 262, 10938-10945] confirm that Trp40 and Trp54 of pIP231a SSB are stacked in the complex, while Trp88 is not. This is the same distribution of stacked Trp residues found in Eco SSB. These results are confirmed further by specific effects observed on the ODMR signals of pIP231a SSB upon binding to poly(Br5U) and poly(dT), which are known to be caused by the stacking of Trp54 with nucleic acid bases.     

Triplet state sublevel kinetics of tryptophan 54 in the complex of Escherichia coli single-stranded DNA binding protein with single-stranded poly(deoxythymidylic) acid.

The individual sublevel kinetics of the lowest triplet state of tryptophan 54 (Trp 54) which is highly perturbed in the complex of Escherichia coli single-stranded DNA binding protein (Eco SSB) with poly(deoxythymidylic) acid (poly[dT]) have been studied by optically detected magnetic resonance (ODMR) spectroscopy. The triplet sublevel decay constants of Trp 54, kx, ky, kz, are 0.99, 0.072, and 0.045 s-1, respectively, in the poly(dT) complex of a point-mutated Eco SSB in which Trp 88 is substituted by phenylalanine. Tx is the only radiative triplet sublevel. Negative polarity of the Tx----Tz and Tx----Ty phosphorescence-detected ODMR signals results from the steady state population pattern, nx greater than ny, nz, and implies that the relations, px greater than or equal to 14py, and px greater than or equal to 22pz exist for the relative populating rates. Spin-orbit coupling between radiative singlet states and the Tx sublevel of the lowest triplet state of Trp 54 is enhanced selectively upon complexing of Eco SSB with poly(dT).     

Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E. coli single-stranded DNA-binding protein and an E. coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.     

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.     

Continuous association of Escherichia coli single-stranded DNA binding protein with stable complexes of recA protein and single-stranded DNA

The single-stranded DNA binding protein of Escherichia coli (SSB) stimulates recA protein promoted DNA strand exchange reactions by promoting and stabilizing the interaction between recA protein and single-stranded DNA (ssDNA). Utilizing the intrinsic tryptophan fluorescence of SSB, an ATP-dependent interaction has been detected between SSB and recA-ssDNA complexes. This interaction is continuous for periods exceeding 1 h under conditions that are optimal for DNA strand exchange. Our data suggest that this interaction does not involve significant displacement of recA protein in the complex by SSB when ATP is present. The properties of this interaction are consistent with the properties of SSB-stabilized recA-ssDNA complexes determined by other methods. The data are incompatible with models in which SSB is displaced after functioning transiently in the formation of recA-ssDNA complexes. A continuous association of SSB with recA-ssDNA complexes may therefore be an important feature of the mechanism by which SSB stimulates recA protein promoted reactions.     

Kinetics of binding of single-stranded DNA binding protein from Escherichia coli to single-stranded nuclei acids

The time course of the reaction of Escherichia coli single-stranded DNA binding protein (E. coli SSB) with poly(dT) and M13mp8 single-stranded DNA has been measured by fluorescence stopped-flow experiments. For poly(dT), the fluorescence traces follow simple bimolecular behavior up to 80% saturation of the polymer with E. coli SSB. A mechanistic explanation of this binding behavior can be given as follows: (1) E. coli SSB is able to translocate very rapidly on the polymer, forming cooperative clusters. (2) In the rate-limiting step of the association reaction, E. coli SSB is bound to the polymer only by one or two of its four contact sites. As compared to poly(dT), association to single-stranded M13mp8 phage DNA is slower by at least 2 orders of magnitude. We attribute this finding to the presence of secondary structure elements (double-stranded structures) in the natural single-stranded DNA. These structures cannot be broken by E. coli SSB in a fast reaction. In order to fulfill its physiological function in reasonable time, E. coli SSB must bind newly formed single-stranded DNA immediately. The protein can, however, bind to such pieces of the newly formed single-stranded DNA which are too short to cover all four binding sites of the E. coli SSB tetramer.     

Energy transfer in complexes of E. coli single-stranded DNA-binding protein with single-stranded poly-(2-thiouridylic acid)

The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR). Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases. Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place. The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U. The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp. The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.     

Stacking interactions of tryptophan residues and nucleotide bases in complexes formed between Escherichia coli single-stranded DNA binding protein and heavy atom-modified poly(uridylic) acid. A study by optically detected magnetic resonance spectroscopy

The complexes formed between Escherichia coli single-stranded DNA binding protein (SSBP) and the heavy atom-modified single-stranded polynucleotides poly(5-BrU) and poly(5-HgU) are investigated using optically detected magnetic resonance (ODMR) methods. In these complexes the triplet state properties of the tryptophan residues are subjected to the external heavy atom effect generated by bromine and mercury atoms and are characterized by a shortened triplet state lifetime and the appearance of the otherwise dark [D] + [E] slow passage ODMR signal. These features provide direct evidence for close range interactions between tryptophan residue(s) and the nucleotide bases in the complexes. The extent of the triplet state lifetime reduction in the case of the SSBP-poly(5-HgU) complex together with steric considerations of the complex structure is consistent only with a van der Waals contact between the perturbed molecule and the heavy atom perturber by means of a stacking interaction. Fast passage ODMR measurements show a lifetime for a sublevel of the perturbed tryptophan chromophore(s) in this complex on the order of 1 ms. The amplitude-modulated phosphorescence microwave double resonance technique captures selectively the broadened and red-shifted phosphorescence spectrum of the heavy atom-perturbed tryptophan residue(s). This work supports a model for the binding of SSBP to single-stranded polynucleotides in which the bases are inserted into hydrophobic regions of the protein, where they are likely to undergo stacking interactions with the indole moiety of buried tryptophan residues.     

Purification and characterization of the single-stranded DNA binding protein from Streptococcus pneumoniae

The Escherichia coli single-stranded DNA binding (SSB) protein is a non-sequence-specific DNA binding protein that functions as an accessory factor for the RecA protein-promoted three-strand exchange reaction. An open reading frame encoding a protein similar in size and sequence to the E. coli SSB protein has been identified in the Streptococcus pneumoniae genome. The open reading frame has been cloned, an overexpression system has been developed, and the protein has been purified to greater than 99% homogeneity. The purified protein binds to ssDNA in a manner similar to that of the E. coli SSB protein. The protein also stimulates the S. pneumoniae RecA protein and E. coli RecA protein-promoted strand exchange reactions to an extent similar to that observed with the E. coli SSB protein. These results indicate that the protein is the S. pneumoniae analog of the E. coli SSB protein. The availability of highly-purified S. pneumoniae SSB protein will facilitate the study of the molecular mechanisms of RecA protein-mediated transformational recombination in S. pneumoniae.     

Limited proteolysis studies on the Escherichia coli single-stranded DNA binding protein. Evidence for a functionally homologous domain in both the Escherichia coli and T4 DNA binding proteins

Limited proteolysis can be used to remove either 42 or 62 amino acids at the COOH terminus of the 18,873-dalton Escherichia coli single-stranded DNA binding protein (SSB). Since poly(dT), but not d(pT)16, increases the rate of this reaction, it appears that cooperative SSB binding to single-stranded DNA (ssDNA) is associated with a conformational change that increases the exposure of the COOH terminus to proteolysis. As a result of this DNA-induced conformational change, we presume that the COOH-terminal region of SSB will become more accessible for interacting with other proteins that utilize the SSB:ssDNA complex as a substrate and that are involved in E. coli DNA replication, repair, and recombination. Removal of this COOH-terminal domain from SSB results in a stronger helix-destabilizing protein which suggests this region may be important for controlling the ability of SSB to denature double-stranded DNA. Since similar results have previously been reported for the bacteriophage T4 gene 32 protein (Williams, K.R., and Konigsberg, W. (1978) J. Biol. Chem. 253, 2463-2470; Hosoda, J., and Moise, H. (1978) J. Biol. Chem. 253, 7547-7555), the acidic, COOH-terminal domains of these two single-stranded DNA binding proteins may be functionally homologous. Preliminary evidence is cited that suggests other prokaryotic and eukaryotic DNA binding proteins may contain similar functional domains essential for controlling their ability to invade double helical DNA.     

Tryptophan 54 and phenylalanine 60 are involved synergistically in the binding of E. coli SSB protein to single-stranded polynucleotides

The binding of both wild-type and point-mutated E. coli single-stranded DNA-binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe-60 is replaced by alanine strongly suggests the participation of Phe-60 in the binding process, possibly by the formation of an extended stacking structure by Trp-54, thymine and Phe-60. This hypothesis is supported by results on the point mutations in which His-55 is replaced by either leucine or tyrosine.     

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.     

Optically detected magnetic resonance of tryptophan residues in complexes formed between a bacterial single-stranded DNA binding protein and heavy atom modified poly(uridylic acid)

Optically detected magnetic resonance (ODMR) methods were employed to study three single-stranded DNA binding (SSB) proteins encoded by plasmids of enteric bacteria: pIP71a, R64, and F. Equilibrium binding isotherms obtained by fluorescence titrations reveal that the complexes of the plasmid SSB proteins with heavy atom modified polynucleotides are readily disrupted by salt. Since all the plasmid SSB proteins show limited solubility at low ionic strength (pIP71a greater than R64 greater than F), we were able to bind only the pIP71a protein to mercurated poly(uridylic acid) [poly(5-HgU)] and brominated poly(uridylic acid) [poly(5-BrU)]. ODMR results reveal the existence of at least one heavy atom perturbed, red-shifted, stacked Trp residue in these complexes. Amplitude-modulated phosphorescence microwave double resonance spectra display selectively the phosphorescence associated with Hg-perturbed Trp residue(s) in the pIP71a SSB protein-poly(5-HgU) complex, which has a broad, red-shifted 0,0-band. Our results suggest that Trp-135 in Escherichia coli SSB, which is absent in the plasmid-encoded SSB proteins, is located in a polar environment and is not involved in stacking interactions with the nucleotide bases. Phosphorescence spectra and lifetime measurements of the pIP71a SSB protein-poly (5-BrU) complex show that at least one Trp residue in the complex does not undergo stacking. This sets a higher limit of two stacking interactions of Trp residues with nucleotide bases in complexes of pIP71a SSB with single-stranded polynucleotides.     

Escherichia coli single-stranded DNA binding protein stimulates the DNA deoxyribophosphodiesterase activity of exonuclease I.

The E. coli single-stranded binding protein (SSB) has been demonstrated in vitro to be involved in a number of replicative, DNA renaturation, and protective functions. It was shown previously that SSB can interact with exonuclease I to stimulate the hydrolysis of single-stranded DNA. We demonstrate here that E. coli SSB can also enhance the DNA deoxyribophosphodiesterase (dRpase) activity of exonuclease I by stimulating the release of 2-deoxyribose-5-phosphate from a DNA substrate containing AP endonuclease-incised AP sites, and the release of 4-hydroxy-2-pentenal-5-phosphate from a DNA substrate containing AP lyase-incised AP sites. E. coli SSB and exonuclease I form a protein complex as demonstrated by Superose 12 gel filtration chromatography. These results suggest that SSB may have an important role in the DNA base excision repair pathway.     

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli.

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.     

Stabilization of recA protein-ssDNA complexes by the single-stranded DNA binding protein of Escherichia coli

In vitro recombination reactions promoted by the recA protein of Escherichia coli are enhanced by the single-stranded DNA binding protein (SSB). SSB affects the assembly of the filamentous complexes between recA protein and ssDNA that are the active form of the recA protein. Here, we present evidence that SSB plays a complex role in maintaining the stability and activity of recA-ssDNA filaments. Results of ATPase, nuclease protection, and DNA strand exchange assays suggest that the continuous presence of SSB is required to maintain the stability of recA-ssDNA complexes under reaction conditions that support their recombination activity. We also report data that indicate that there is a functional distinction between the species of SSB present at 10 mM magnesium chloride, which enhances recA-ssDNA binding, and a species present at 1 mM magnesium chloride, which displaces recA protein from ssDNA. These results are discussed in the context of current models of SSB conformation and of SSB action in recombination activities of the recA protein.     

p10, a low molecular weight single-stranded nucleic acid binding protein of murine leukemia retroviruses, shows stacking interactions of its single tryptophan residue with nucleotide bases

Room temperature fluorescence and low-temperature phosphorescence studies of the association of p10, a basic low molecular weight single-stranded DNA binding protein isolated from murine leukemia viruses, point to the involvement of its single tryptophan residue in a close-range interaction with single-stranded polynucleotides. Optically detected triplet-state magnetic resonance (ODMR) techniques applied to the complex of p10 protein with the heavy atom derivatized polynucleotide poly(5-HgU) demonstrate the occurrence of stacking interactions of Trp35 with nucleic acid bases, thus agreeing with earlier reports that this residue is involved in the binding process [Karpel, R. L., Henderson, L. E., & Oroszlan, S. (1987) J. Biol. Chem. 262, 4961-4967].     

A single-stranded DNA-binding protein promotes the binding of the purified oestrogen receptor to its responsive element.

The purified human oestrogen receptor (hER) does not form a detectable complex with an oestrogen responsive element (ERE) under conditions where hER-ERE complexes are readily formed with crude extracts from Hela or yeast cells expressing the hER. This indicates that other factor(s) are necessary for ER-ERE binding. Such a ER DNA binding stimulatory factor (DBSF) has been purified from the yeast Saccharomyces cerevisiae. It is a 45 kDa single-stranded DNA-binding protein (SSB) which cannot be substituted for by the purified E. coli SSB.