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T M Lohman

Publications and source records attributed to T M Lohman.

At least 73 records · Page 4Linked to original sources
PubMed1990

Escherichia coli helicase II (UvrD) protein initiates DNA unwinding at nicks and blunt ends.

The Escherichia coli uvrD gene product, helicase II, is required for both methyl-directed mismatch and uvrABC excision repair and is believed to function by unwinding duplex DNA. Initiation of unwinding may occur specifically at either a mismatch or a nick, although no direct evidence for this has previously been reported. It has recently been shown that helicase II can unwind fully duplex linear and nicked circular DNA with lengths of at least approximately 2700 base pairs in vitro; hence, a flanking region of single-stranded DNA is not required to initiate DNA unwinding. In studies with uniquely nicked duplex DNA, we present EM evidence that helicase II protein initiates DNA unwinding at the nick, with unwinding proceeding bidirectionally. We also show that helicase II protein initiates DNA unwinding at the blunt ends of linear DNA, rather than in internal regions. These data provide direct evidence that helicase II protein can initiate unwinding of duplex DNA at a nick, in the absence of auxiliary proteins. We propose that helicase II may initiate unwinding from a nick in a number of DNA repair processes.

Adenosine Triphosphatases↗
PubMed1990

Thermodynamic extent of counterion release upon binding oligolysines to single-stranded nucleic acids.

A major contribution to the binding free energy associated with most protein-nucleic acid complexes is the increase in entropy due to counterion release from the nucleic acid that results from electrostatic interactions. To examine this quantitatively, we have measured the thermodynamic extent of counterion release that results from the interaction between single-stranded homopolynucleotides and a series of oligolysines, possessing net charges z = 2-6, 8, and 10. This was accomplished by measuring the salt dependence of the intrinsic equilibrium binding constants--i.e., (delta log Kobs/delta log[K+])--over the range from 6 mM to 0.5 M potassium acetate. These data provide a rigorous test of linear polyelectrolyte theories that have been used to interpret the effects of changes in bulk salt concentration on protein-DNA binding equilibria, since single-stranded nucleic acids have a lower axial charge density than duplex DNA. Upon binding to poly(U), the thermodynamic extent of counterion release per oligolysine charge, z, is 0.71 +/- 0.03, which is significantly less than unity and less than that measured upon binding duplex DNA. These results are most simply interpreted using the limiting law predictions of counterion condensation and cylindrical Poisson-Boltzmann theories, even at the high salt concentrations used in our experiments. Accurate estimates of the thermodynamic extent of counterion binding and release for model systems such as these facilitate our understanding of the energetics of protein-nucleic acid interactions. These data indicate that for simple oligovalent cations, the number of ionic interactions formed in a complex with a linear nucleic acid can be accurately estimated from a measure of the salt dependence of the equilibrium binding constant, if the thermodynamic extent of ion release is known.

Kinetics↗
PubMed1989

Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA.

We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1989

Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of DNA is highly specific for duplex DNA.

We show that iron(II)-EDTA-catalyzed cleavage of duplex DNA is much more rapid than cleavage of single-stranded DNA that does not form intramolecular base pairs. Comparisons of the extent of cleavage of the fully single-stranded oligonucleotides d(pT)70, d(pA)70, and d(pC)35 and the duplex DNA d(pT)70.d(pA)70 indicate that the extent of cleavage increases significantly upon formation of the duplex structure. These observations indicate that accessibility of the DNA sugars to the presumed cleaving agent, hydroxyl radical, is not the major determinant for cleavage and that most likely a direct interaction between Fe(II) and the DNA is required. As a result, the interpretation of DNA cleavage experiments performed with this reagent to obtain detailed structural information should be pursued with caution until the mechanism of the cleavage reaction is better understood.

DNA↗
PubMed1989

Large-scale purification and characterization of the Escherichia coli rep gene product.

We report a procedure for the large-scale purification of the Escherichia coli Rep protein, a helicase that is involved in the replication of the E. coli chromosome as well as a number of single-stranded bacteriophages. The procedure starts with E. coli cells harboring an overproducing plasmid, pRepO, in which the E. coli rep gene is under transcriptional control of the inducible lambda PL promoter (Colasanti, J., and Denhardt, D. T. (1987) Mol. Gen. Genet. 209, 382-390). The purification procedure results in greater than 98% pure Rep protein, which is free of contaminating nuclease activity, with yields of 40-50 mg of Rep protein/50 g of induced MZ-1/pRepO cells. We also show that cell death occurs upon inducing such a large overproduction of the E. coli Rep protein in MZ-1/pRepO. The Rep protein purified by this procedure has high specific single-stranded DNA-dependent ATPase activity, as well as helicase activity, with an apparent 3' to 5' directionality. The extinction coefficient of purified E. coli Rep protein is epsilon 280 = 1.16 +/- 0.04 ml mg-1 cm-1 (8.47 +/- 0.28 X 10(4) M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25 degrees C. The solubility properties of the purified Rep protein have been examined as a function of glycerol, NaCl, MgCl2, ATP, and ADP concentrations at 25 and 37 degrees C (pH 7.5). Rep protein solubility decreases significantly with decreasing concentrations of glycerol and monovalent salt and increasing temperature; however, the presence of 1.5 mM ATP or ADP or MgCl2 at low NaCl concentrations increases the solubility. At 4 degrees C, in the presence of 20% glycerol and greater than or equal to 50 mM NaCl, the free Rep protein exists as a stable monomer under all conditions examined (+/- ATP and +/- MgCl2). The single-stranded DNA-dependent ATPase activity decreases with increasing glycerol concentration, such that in 25% (v/v) glycerol it has approximately 40% of its activity as compared to solutions that contain no glycerol. The dependence of the single-stranded DNA-dependent ATPase activity on salt concentration for a series of monovalent salts indicates the presence of both cation and anion effects, with decreasing activity in the order glutamate greater than acetate greater than chloride. The ability to obtain highly purified E. coli Rep protein in large quantities with relative ease will greatly facilitate physical characterizations of the protein and its interactions with DNA.

Adenosine Triphosphatases↗
PubMed1989

Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. I. Evidence and a quantitative model.

The interaction of the Escherichia coli single strand binding (SSB) protein with single-stranded DNA is complex, since a number of different binding modes have been observed, with different DNA site sizes and binding properties and the transitions among these binding modes are strongly influenced by solution conditions in vitro. Recent experiments have suggested the existence of negative co-operativity among the multiple DNA binding sites within individual SSB tetramers. In order to probe this negative co-operativity, we have examined the binding of a series of oligonucleotides of varying length, using the quenching of the intrinsic SSB protein fluorescence to monitor binding. The stoichiometries for saturation of the SSB tetramer are 4, 2, 2, 1 and 1, for the oligonucleotides, dT(pT)N-1, with N = 16, 28, 35, 56 and 70, respectively, indicating that one molecule of either dT(pT)27 or dT(pT)34 interacts with two SSB subunits, whereas one molecule of dT(pT)15 interacts with only a single subunit. Saturation of the SSB tetramer with dT(pT)15, dT(pT)34, dT(pT)69 or poly(dT) results in 85 to 90% quenching of the SSB fluorescence, whereas saturation with dT(pT)27 or dT(pT)55 results in only 80% and 72% quenching, respectively. Therefore, a single-stranded DNA of at least 64 nucleotides is required to wrap around an SSB tetramer fully and interact with all four subunits. A quenching of 50(+/- 2)% is observed upon filling only half of the subunits with either one molecule of dT(pT)34 or two molecules of dT(pT)15, which agrees with the quenching and site size observed in the (SSB)35 polynucleotide binding mode. Direct binding measurements indicate that the binding of dT(pT)27 to its second site is influenced by the oligonucleotide that occupies the first binding site (either dT(pT)27 or dT(pT)34), providing proof for the existence of a true negative co-operativity. This negative co-operativity is observed also for the binding of the shorter oligonucleotide, dT(pT)15. A statistical thermodynamic ("square") model gives an excellent description of the binding of all oligonucleotides possessing multiple sites on the SSB tetramer, based on only two interaction constants, the intrinsic binding constant, KN, and the negative co-operativity parameter, sigma N. These data indicate that the binding sites (subunits) on the unliganded SSB tetramer are all equivalent, but that a non-equivalence between dimers of subunits within the tetramer is induced upon binding ssDNA.

Bacterial Proteins↗
PubMed1989

Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects.

We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant KN, and the negative co-operativity constant, sigma N. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. delta log KN/delta log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that delta Ho = -26(+/- 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. sigma N less than 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of sigma 16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for sigma 16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.

Bacterial Proteins↗
PubMed1989

On the cooperative binding of large ligands to a one-dimensional homogeneous lattice: the generalized three-state lattice model.

We present the general secular equation for three-state lattice models for the cooperative binding of large ligands to a one-dimensional lattice. In addition, a closed-form expression for the isotherm is also obtained, that can be used with all values of the cooperativity parameter omega(0 less than omega less than infinity) thus eliminating the need for multiple equations.

Chemical Phenomena↗
PubMed1988

Negative cooperativity within individual tetramers of Escherichia coli single strand binding protein is responsible for the transition between the (SSB)35 and (SSB)56 DNA binding modes.

We have examined the binding of the oligonucleotide dT (pT)34 to the Escherichia coli SSB protein as a function of NaCl and MgCl2 concentration (25 degrees C, pH 8.1) by monitoring the quenching of the intrinsic protein fluorescence. We find two binding sites for dT(pT)34 per single strand binding (SSB) protein tetramer, with each site possessing widely different affinities depending on the salt concentration. At 200 mM NaCl, we observe nearly stoichiometric binding of dT(pT)34 to both binding sites within the SSB tetramer, although a difference in the affinities is still apparent. However, when the NaCl concentration is lowered, the overall affinity of dT(pT)34 for the second site on the SSB tetramer decreases dramatically. At 1.5 mM NaCl, only a single molecule of dT(pT)34 can bind per SSB tetramer, even with a 10-fold molar excess of dT(pT)34. MgCl2 is effective at 100-fold lower concentrations than NaCl in promoting the binding of the second molecule of dT(pT)34. This binding behavior reflects an intrinsic property of the SSb tetramer, since it is also observed upon binding of smaller oligonucleotides, and the simplest explanation is that a salt-dependent negative cooperativity exists between DNA binding sites within the SSB tetramer. This phenomenon is also responsible for the transition between the two SSB-single strand (ss) polynucleotide binding modes that cover 35 and 56 nucleotides per tetramer [Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802]. Extreme negative cooperativity stabilizes the (SSB)35 binding mode, in which the SSB tetramer binds tightly to ss DNA with only two of its subunits while the other two subunits remain unligated.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1988

Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects.

We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single-stranded DNA (Lohman, T. M. & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W. & Lohman, T. M. (1986) Biochemistry 25, 7799-7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes. "Reverse" titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25 degrees C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgCl2 concentrations. Under all conditions in which "reverse" titrations were performed, we observe three distinct binding modes with site sizes of 35 +/- 2, 56 +/- 3, and 65 +/- 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH. A net uptake of both cations and anions accompanies the transitions from the (SSB)35 to the (SSB)56 binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs. The transition from the (SSB)56 to the (SSB)65 binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25 degrees C), and a net uptake of both cations and anions accompanies this transition. We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl2 concentration (20 degrees C, pH 8.1) and observe an increase in s20,w, which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations. The frictional coefficient of the complex decreases by a factor of two in progressing from the (SSB)35 to the (SSB)65 binding mode, indicating a progressive compaction of the complex throughout the transition. The transition between the (SSB)35 and the (SSB)56 complex is dependent on the protein binding density, with the lower site size (SSB)35 complex favored at higher binding density. These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked.(ABSTRACT TRUNCATED AT 400 WORDS)

Anions↗
PubMed1988

Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificity.

The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(dA), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)65 binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model ("tetramer/octamer" model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, Kobsd, and cooperativity parameter, omegoT/O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)65 binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of omegaT/O = 410 +/- 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25 degrees C, pH 8.1). However, Kobsd decreases steeply with increasing salt concentration, such that alpha log Kobsd/alpha log [NaCl] = -7.4 +/- 0.5 for poly(U), -6.1 +/- 0.6 for poly(dA), and -6.2 +/- 0.3 for poly(A) (25.0 degrees C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure alpha log Kobsd/alpha log [NaBr] = -5.7 +/- 0.7, with a lower value of omega T/O = 130 +/- 70. The polynucleotide specificity of the (SSB)65 binding mode (0.20 M NaCl, 25.0 degrees C, pH 8.1) is Kobsd(dT) greater than Kobsd(dC) much greater than Kobsd(ss M13 DNA) greater than Kobsd(I) greater than Kobsd(U) = 8Kobsd(dA) = 87Kobsd(A) much greater than Kobsd(C).(ABSTRACT TRUNCATED AT 400 WORDS)

Anions↗
PubMed1987

Limited co-operativity in protein-nucleic acid interactions. A thermodynamic model for the interactions of Escherichia coli single strand binding protein with single-stranded nucleic acids in the "beaded", (SSB)65 mode.

We present a statistical thermodynamic model ("tetramer/octamer" model) that describes the equilibrium binding of the Escherichia coli single strand binding (SSB) protein to single-stranded nucleic acids in its "beaded" binding mode, which seems to be equivalent to the high site size, (SSB)65 binding mode. The method of sequence-generating functions is used to derive the model, which accounts for the observation that clustering of bound SSB tetramers is limited to the formation of octamers, which have been observed as "beads" in the electron microscope. The model also accounts for the overlap of potential protein binding sites on the nucleic acid. The "tetramer/octamer" model is fully described by only three parameters: the site size, n; the intrinsic equilibrium constant, K; and the co-operativity parameter, omega, and we obtain exact, closed form expressions for the binding isotherm as well as the distribution of DNA-bound SSB tetramers and octamers. The closed form expressions allow one to calculate easily average binding properties and analyze experimental binding isotherms to obtain estimates of K and omega. In order to test the tetramer/octamer model, we have determined the equilibrium binding isotherm for the E. coli SSB protein-poly(U) interaction in 0.2 M-NaCl over a wide range of binding densities. These are conditions in which the low co-operativity (SSB)65 binding mode solely exists. The tetramer/octamer model provides a much better description of the experimental isotherm over the entire binding density range than a model that assumes the formation of clusters of unlimited size. A co-operativity parameter of omega = 420 +/- 80 provides a good fit to data for SSB binding to poly(dA) and poly(U), corresponding to an interaction free energy of -3.6 kcal/mol of SSB octamer formed. On the basis of this moderate value of omega, the tetramer/octamer model predicts that at low to intermediate binding densities, a significant fraction of bound SSB exists in the form of tetramers co-existing with octamers. In the case of E. coli SSB protein binding in the "beaded", (SSB)65 mode this model provides a significant improvement over previous treatments which assume unlimited nearest-neighbor interactions, since the binding parameters, K and omega, represent physically meaningful interaction constants rather than fitting parameters.

Allosteric Regulation↗
PubMed1987

A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions.

We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand. This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand. Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid. We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein. With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT. For E. coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Chrysogelos, S., & Griffith, J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5803], we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.

Bacterial Proteins↗
PubMed1986

Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA.

Four distinct binding modes for the interaction of Escherichia coli single-strand binding (SSB) protein with single-stranded (ss) DNA have been identified on the basis of quantitative titrations that monitor the quenching of the SSB protein fluorescence upon binding to the homopolynucleotide poly(dT) over a range of MgCl2 and NaCl concentrations at 25 and 37 degrees C. This is the first observation of multiple binding modes for a single protein binding to DNA. These results extend previous studies performed in NaCl (25 degrees C, pH 8.1), in which two distinct SSB-ss DNA binding modes possessing site sizes of 33 and 65 nucleotides per bound SSB tetramer were observed [Lohman, T.M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603]. Each of these binding modes differs in the number of nucleotides occluded upon interaction with ss DNA (i.e., site size). Along with the previously observed modes with site sizes of 35 +/- 2 and 65 +/- 3 nucleotides per tetramer, a third distinct binding mode, at 25 degrees C, has been identified, possessing a site size of 56 +/- 3 nucleotides per bound SSB tetramer, which is stable over a wide range of MgCl2 concentrations. At 37 degrees C, a fourth binding mode is observed, possessing a site size of 40 +/- 2 nucleotides per tetramer, although this mode is observable only over a small range of salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Single-Stranded↗
PubMed1986

Salt-dependent changes in the DNA binding co-operativity of Escherichia coli single strand binding protein.

The co-operative nature of the binding of the Escherichia coli single strand binding protein (SSB) to single-stranded nucleic acids has been examined over a range of salt concentrations (NaCl and MgCl2) to determine if different degrees of binding co-operativity are associated with the two SSB binding modes that have been identified recently. Quantitative estimates of the binding properties, including the co-operativity parameter, omega, of SSB to single-stranded DNA and RNA homopolynucleotides have been obtained from equilibrium binding isotherms, at high salt (greater than or equal to 0.2 M-NaCl), by monitoring the fluorescence quenching of the SSB upon binding. Under these high salt conditions, where only the high site size SSB binding mode exists (65 +/- 5 nucleotides per tetramer), we find only moderate co-operativity for SSB binding to both DNA and RNA, (omega = 50 +/- 10), independent of the concentration of salt. This value for omega is much lower than most previous estimates. At lower concentrations of NaCl, where the low site size SSB binding mode (33 +/- 3 nucleotides/tetramer) exists, but where SSB affinity for single-stranded DNA is too high to estimate co-operativity from classical binding isotherms, we have used an agarose gel electrophoresis technique to qualitatively examine SSB co-operativity with single-stranded (ss) M13 phage DNA. The apparent binding co-operativity increases dramatically below 0.20 M-NaCl, as judged by the extremely non-random distribution of SSB among the ssM13 DNA population at low SSB to DNA ratios. However, the highly co-operative complexes are not at equilibrium at low SSB/DNA binding densities, but are formed only transiently when SSB and ssDNA are directly mixed at low concentrations of NaCl. The conversions of these metastable, highly co-operative SSB-ssDNA complexes to their equilibrium, low co-operativity form is very slow at low concentrations of NaCl. At equilibrium, the SSB-ssDNA complexes seem to possess the same low degree of co-operativity (omega = 50 +/- 10) under all conditions tested. However, the highly co-operative mode of SSB binding, although metastable, may be important during non-equilibrium processes such as DNA replication. The possible relation between the two SSB binding modes, which differ in site size by a factor of two, and the high and low co-operativity complexes, which we report here, is discussed.

Allosteric Regulation↗
PubMed1986

Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control.

We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81-93], carrying the bacteriophage lambda PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor. After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste. In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein.

Bacteriophage lambda↗