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T M Lohman

Publications and source records attributed to T M Lohman.

At least 37 records · Page 2Linked to original sources
PubMed1996

ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: implications for ATP driven helicase translocation.

To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16. Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation. This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer. However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency). These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA. Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.

Adenosine Triphosphatases↗
PubMed1996

ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states.

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992), Science 256, 350-355]. THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C). The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.

Adenosine Triphosphatases↗
PubMed1996

A highly salt-dependent enthalpy change for Escherichia coli SSB protein-nucleic acid binding due to ion-protein interactions.

We have examined the linkage between salt concentration and temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-drive; however, the value of [symbol see text] log Kobs/ [symbol see text] log [NaCl] is highly temperature- dependent, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 degrees C. This indicates that delta Hobs for SSB-poly(U) binding is strongly dependent on [NaCl]; based on van't Hoff analyses, delta Hobs varies from -57 +/- 3 kcal/mol at 0.18 M NaCl to -34 +/- 3 kcal/mol at 042 M NaCl ([symbol see text] delta Hobs/[symbol see text] log [NaCl] = 60 +/- 5 kcal/mol). However, [symbol see text] delta Hobs/[symbol see text] log [NaF] is independent of temperature (25-37 degrees C), indicating that the effect of [NaCl] on delta Hobs is due primarily to Cl-. Similar effects were also observed for SSB binding to dA(pA)69. We also measured delta Hobs and its dependence on [NaCl] for SSB binding dT(pT)69 by ITC and find delta Hobs = -144 +/- 4 kcal/mol (0.175 M NaCl, pH 8.1, 25 degrees C) and [symbol see text] delta Hobs/ [symbol see text] log [NaCl] = 46 +/- 2 kcal/ mol (0.175-2.0 M NaCl). These large effects of [NaCl] on delta Hobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with delta H > > 0. Effects of salt concentration on delta Hobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ of K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such salt effects on delta Hobs may serve as a signature for differential ion-protein binding. These results underscore the need to examine the linkage of [salt] to delta Hobs, as well as delta Hobs degrees and delta S(obs) degrees, in order to understand the bases for stability and specificity of protein-nucleic acid interactions.

Bacterial Proteins↗
PubMed1996

Large electrostatic differences in the binding thermodynamics of a cationic peptide to oligomeric and polymeric DNA.

Results presented here demonstrate that the thermodynamics of oligocation binding to polymeric and oligomeric DNA are not equivalent because of long-range electrostatic effects. At physiological cation concentrations (0.1-0.3 M) the binding of an oligolysine octacation KWK6-NH2 (+8 charge) to single-stranded poly(dT) is much stronger per site and significantly more salt concentration dependent than the binding of the same ligand to an oligonucleotide, dT(pdT)10 (-10 charge). These large differences are consistent with Poisson-Boltzmann calculations for a model that characterizes the charge distributions with key preaveraged structural parameters. Therefore, both the experimental and the theoretical results presented here show that the polyelectrolyte character of a polymeric nucleic acid makes a large contribution to both the magnitude and the salt concentration dependence of its binding interactions with simple oligocationic ligands.

Cations↗
PubMed1996

Kinetic mechanism of DNA binding and DNA-induced dimerization of the Escherichia coli Rep helicase.

The monomeric Escherichia coli Rep protein undergoes a DNA-induced dimerization upon binding either single-stranded (ss) or duplex DNA with the dimer being the active form of the Rep helicase. Using stopped-flow fluorescence, we have determined a minimal kinetic mechanism for this reaction in which Rep monomer (P) binds to ss oligodeoxynucleotides (dN(pN)15) (S) by a two-step mechanism to form PS*, which can then dimerize with P to form P2S as indicated: [reaction in text]. This minimal mechanism is supported by four independent studies in which the kinetics were monitored by changes in fluorescence intensity of three different probes: the intrinsic Rep tryptophan fluorescence, the fluorescence of d(T5(2-AP)T4(2-AP)T5), containing the fluorescent base, 2-aminopurine (2-AP), and dT(pT)15 labeled at its 3'-end with fluorescein (3'-F-dT(pT)15). Simultaneous (global) analysis of the time courses of d(T5(2-AP)T4(2-AP)T5) (100 nM) binding to a range of Rep monomer concentrations (25-400 nM) yields the following rate constants: k1 = (3.3 +/- 0.5) x 10(7) M-1 s-1; k-1 = 1.4 +/- 0.4 s-1; k2 = 2.7 +/- 0.9 s-1; k-2 = 0.21 +/- 0.06 s-1; k3 = (4.5 +/- 0.3) x 10(5) M-1 s-1; k-3 = 0.0027 +/- 0.0008 s-1 [20 mM Tris-HCl, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4.0 degrees C]. This mechanism provides direct evidence that Rep monomers can bind ss DNA and that ss DNA binding induces a conformational change in the Rep monomer that is probably required for Rep dimerization. This conformational change is likely to be large and global since it is detected by all three fluorescence probes. The apparent bimolecular rate constant for Rep monomer binding to 3'-F-dT(pT)15 [k1(app) = (6.0 +/- 0.7) x 10(7) M-1 s-1] is slightly larger than measured with d(T5(2-AP)T4(2-AP)T5) binding. The apparent rate constant for dissociation of d(T5(2-AP)T4(2-AP)T5) (S) from the half-ligated Rep dimer, P2S, increases with increasing concentration of a nonfluorescent competitor ss DNA (d(T5-AT4AT5)) (C), indicating transient formation of a doubly ligated P2SC intermediate. However, the apparent bimolecular rate constant for binding of C to P2S is extremely slow (> or = 250 M-1 s-1), suggesting the occurrence of a multistep process before dissociation of ss DNA. In the absence of competitor DNA, dissociation of ss DNA from P2S occurs only after slow dissociation of the Rep dimer to form PS* + P. The implications of these results for Rep-catalyzed DNA unwinding are discussed.

Adenosine Triphosphatases↗
PubMed1996

Mechanisms of helicase-catalyzed DNA unwinding.

DNA helicases are essential motor proteins that function to unwind duplex DNA to yield the transient single-stranded DNA intermediates required for replication, recombination, and repair. These enzymes unwind duplex DNA and translocate along DNA in reactions that are coupled to the binding and hydrolysis of 5'-nucleoside triphosphates (NTP). Although these enzymes are essential for DNA metabolism, the molecular details of their mechanisms are only beginning to emerge. This review discusses mechanistic aspects of helicase-catalyzed DNA unwinding and translocation with a focus on energetic (thermodynamic), kinetic, and structural studies of the few DNA helicases for which such information is available. Recent studies of DNA and NTP binding and DNA unwinding by the Escherichia coli (E. coli) Rep helicase suggest that the Rep helicase dimer unwinds DNA by an active, rolling mechanism. In fact, DNA helicases appear to be generally oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites. The apparent mechanistic similarities and differences among these DNA helicases are discussed.

Biological Transport↗
PubMed1995

Thermodynamics of charged oligopeptide-heparin interactions.

To better understand the electrostatic component of the interaction between proteins and the polyanion heparin, we have investigated the thermodynamics of heparin binding to positively charged oligopeptides containing lysine or arginine and tryptophan (KWK-CO2 and RWR-CO2). The binding of these peptides to heparin is accompanied by an enhancement of the peptide tryptophan fluorescence, and we have used this to determine equilibrium binding constants. The extent of fluorescence enhancement is similar for both peptides, suggesting that the tryptophan interaction is similar for both. Titrations of these peptides with a series of simple salts suggest that this fluorescence enhancement is due to the interaction of tryptophan with sulfate moieties on the heparin. Equilibrium association constants, Kobs (M-1), for each peptide binding to heparin were measured as a function of temperature and monovalent salt concentration in the limit of low peptide binding density. At pH 6.0, 25 degrees C, 20 mM KCH3CO2, Kobs = 3.2 (+/- 0.3) x 10(3) M-1 for KWK-CO2 binding, whereas Kobs = 4.5 (+/- 0.5) x 10(3) M-1 for RWR-CO2. However, the dependence of Kobs on KCH3CO2 concentration is the same for both oligopeptides, each of which possesses a net charge of +2 at pH 6.0. The logarithm of Kobs is a linear function of the logarithm of [KCH3CO2] over the range from 12 mM < or = KCH3CO2 < or = 30 mM (pH 6.0, 25 degrees C), with (delta log Kobs/delta log [KCH3CO2]) = -2.0 +/- 0.3, indicating that approximately 2 ions are released per bound peptide upon formation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Helicase-catalyzed DNA unwinding: energy coupling by DNA motor proteins.

DNA helicases catalyze the unwinding of double-stranded (ds) DNA to yield the single-stranded (ss) DNA intermediates required in DNA replication, recombination, and repair. DNA helicases couple the free energy of nucleoside triphosphate (NTP) binding and hydrolysis to separate the two complementary DNA strands while also translocating vectorially along the DNA substrate. As such, helicases are functionally DNA motor proteins. The functional form of helicases generally appears to be oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites that are required for translocation and DNA unwinding. The affinity of ss- versus dsDNA for these multiple DNA binding sites is modulated allosterically by NTP binding, hydrolysis, and product release, which is central to helicase-catalyzed DNA unwinding. The mechanistic details of the DNA unwinding, translocation, and NTPase reactions are only starting to emerge. We discuss energy coupling by DNA helicases in general, and by the dimeric E. coli Rep helicase in particular, focusing on the similarities of these enzymes to classical motor proteins.

Adenosine Triphosphatases↗
PubMed1994

Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 1. Use of fluorescent nucleotide analogues.

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis. The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the functionally active form of the Rep helicase. As a first step toward understanding how ATP binding and hydrolysis are coupled energetically to DNA unwinding, we have investigated the kinetic mechanism of nucleotide binding to the Rep monomer (P) using stopped-flow techniques and the fluorescent ATP analogue, 2'(3')-O-(N-methylanthraniloyl-ATP (mantATP). The fluorescence of mantATP is enhanced upon Rep binding due to energy transfer from tryptophan. The results are consistent with the following two-step mechanism, in which the bimolecular association step is followed by a conformational change in the P-mantATP complex: P + mantATP [formula: see text] P-mantATP [formula: see text] (P-mantATP). The following rate and equilibrium constants were determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 5 mM MgCl2, and 10% (v/v) glycerol: k+1 = (1.1 +/- 0.2) x 10(7) M-1 s-1; k-1 = 3.2 (+/- 0.5) s-1; k+2 = 2.9 (+/- 0.5) s-1; k-2 = 0.04 (+/- 0.005) s-1; K1 = k+1/k-1 = (3.4 +/- 0.8) x 10(6) M-1; K2 = k+2/k-2 = 73 (+/- 10); Koverall = K1K2 = (2.30 +/- 0.6) x 10(8) M-1. Similar rate and equilibrium constants are obtained with mantATP gamma S, whereas the apparent rate constant for mantAMPPNP binding is 15-fold lower than for mantATP and equilibrium binding is weaker (Koverall approximately 10(6) M-1). Rep monomer does bind mantATP in the absence of Mg2+ (Koverall approximately 5 x 10(5) M-1), although the four rate constants in the above reaction increase by at least 8-fold (k-1 and k-2 increase by approximately 100- and approximately 1000-fold, respectively). The affinities of Mg2+ for P-mantATP and (P-mantATP)* are 10- and 1000-fold higher than those for nucleotide-free Rep monomer, indicating that the second step in the reaction is associated with a marked increase in Mg2+ affinity. The bound Mg2+ in a (P-mantATP)*-Mg2+ complex dissociates at a rate that is comparable to the rate of mantATP release.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1994

Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 2. Application of a kinetic competition approach.

The Escherichia coli Rep protein is a DNA helicase that functions as a homodimer to catalyze the unwinding of duplex DNA during DNA replication in a reaction that is coupled to the binding and hydrolysis of ATP. As a first step toward a molecular understanding of the interactions of Rep with adenine nucleotides, we have investigated the kinetic mechanism of adenine nucleotide binding to the Rep monomer, which is the state of the protein in the absence of DNA. Although ATP binding to Rep does not significantly change the intrinsic tryptophan fluorescence, the binding of the fluorescent nucleotide analogue, 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) is associated with a large increase in mant nucleotide fluorescence intensity [lambda ex = 290 nm, lambda em > 420 nm; Moore, K. J. M., & Lohman, T. M. (1994) Biochemistry (preceding article in this issue)]. We have used the fluorescence signal from mantATP binding to monitor the kinetics of nonfluorescent nucleotide binding to Rep by a kinetic competition approach. The simultaneous and parallel binding of a mixture of mantATP and ATP to the Rep monomer is associated with a complex triphasic fluorescence transient during the approach to equilibrium. Global analysis of the fluorescence transients over a range of [ATP] by numerical integration techniques was used to define the kinetic mechanism of ATP binding and to determine the elementary rate constants. Using this approach, the kinetic rate constants for ADP, ATP gamma S, AMPPNP, AMP, adenosine, and inorganic phosphate were also determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 10% (v/v) glycerol, and 5 mM MgCl2. The kinetics of adenine nucleotide binding to the Rep monomer are similar to those observed with the mant nucleotides under identical experimental conditions (Moore & Lohman, 1994). The kinetic competition data are consistent with the following two-step mechanism for the binding of ATP, ADP, and ATP gamma S, where P is the Rep monomer and A is the adenine nucleotide: P+A [formula: see text] P-A [formula: see text] (P-A). In the presence of 5 mM MgCl2, the values of K+1 (approximately 10(7) M-1 s-1) and k+2 (approximately 10 s-1) are comparable for each nucleotide, whereas k+2 > k-1 for ATP and ATP gamma S while for ADP k+2 << k-1; hence, differences in the overall equilibrium binding affinities of these nucleotides are primarily due to changes in k-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1994

Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer.

We describe a fluorescence assay that can be used to monitor helicase-catalyzed unwinding of duplex DNA continuously in real time. The assay is based on the observation that fluorescence resonance energy transfer (FRET) occurs between donor (fluorescein) and acceptor (hexachlorofluorescein) fluorophores that are in close proximity due to their covalent attachment to the 3' and 5' ends of the complementary strands of a duplex oligodeoxynucleotide. FRET results in a reduction in the fluorescence emission intensity of fluorescein in the duplex DNA substrate relative to that observed for fluorescein-labeled single stranded DNA. Therefore, an enhancement of fluorescein fluorescence (lambda ex = 492 nm; lambda em = 520 nm) occurs upon helicase-catalyzed unwinding of the duplex DNA and separation of the complementary strands. The fluorescence assay is extremely sensitive, allowing DNA unwinding reactions to be monitored continuously at DNA concentrations as low as 1 nM in a fluorescence stopped-flow experiment. We demonstrate the use of this DNA substrate in pre-steady state, single turnover studies of duplex DNA unwinding catalyzed by the Escherichia coli Rep helicase, monitored by fluorescence stopped flow. We show that the fluorescence enhancement monitors Rep-catalyzed DNA unwinding by comparisons with identical kinetic studies carried out using rapid chemical quench-flow techniques. Single turnover kinetic studies performed at 1 nM DNA as a function of excess Rep concentration show that Rep-catalyzed unwinding of an 18 base pair duplex containing a 3'-ss-(dT)20 tail is biphasic and can be described by the sum of two exponential terms. The observed rate constant of the first phase is independent of [Rep] (20-300 nM) and measures the rapid single turnover, unwinding of the duplex DNA by Rep dimers bound in productive complexes (1.3 +/- 0.2 s-1; 23 +/- 3 base pairs s-1 at 25.0 degrees C). The observed rate constant for the second phase increases linearly with [Rep], reflecting DNA unwinding that is limited by a Rep binding event occurring with a bimolecular rate constant of (1.8 +/- 0.1) x 10(5) M-1 s-1, which may reflect the rate constant for Rep dimerization on DNA. Kinetic competition studies indicate that both Rep subunits are bound stably to the DNA substrate in the productive complex that is unwound in the fast phase. The results of these kinetic studies are consistent with an active, rolling mechanism for Rep-catalyzed unwinding of DNA [Wong, I., & Lohman, T. M., (1992) Science 256, 350].(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1994

Apparent heat capacity change accompanying a nonspecific protein-DNA interaction. Escherichia coli SSB tetramer binding to oligodeoxyadenylates.

We have examined the effects of temperature on the equilibrium constant, Kobs, for Escherichia coli SSB tetramer binding to a series of single-stranded (ss) oligodeoxyribonucleotides, dT(pT)n, dC(pC)n, and dA(pA)n (n = 34, 55, and 69) in order to investigate the thermodynamic basis for the strong preference of E. coli SSB (and other SSB proteins) for binding polypyrimidine stretches of ss-DNA. In addition to the expected base-dependent differences in the magnitude of Kobs, we also observe qualitatively different temperature dependencies for the binding of the SSB tetramer to oligodeoxyadenylates. Linear van't Hoff plots are obtained for SSB tetramer binding to dT(pT)n and dC(pC)n, with delta H0obs ranging from -50 to -100 kcal/mol depending on the oligodeoxynucleotide length and salt concentration. In contrast, all van't Hoff plots for SSB tetramer binding to dA(pA)N are distinctly nonlinear with maxima in K(obs) occurring near 25 degrees C, indicative of an apparent large negative change in molar heat capacity (delta C0P,obs < 0). Thus for the SSB-dA(pA)n interaction, delta H0obs and delta S0obs are both highly temperature dependent, but compensate such that delta G0obs is relatively insensitive to temperature. These nonlinear nonlinear van't Hoff plots are not due to coupling of SSB assembly to dA(pA)n binding or to temperature-dependent shifts in the formation of other SSB-DNA binding modes. The nonlinear van't Hoff plots for SSB tetramer binding to dA(pA)n appear to result from the coupling of two processes: (1) the unstacking of the dA(pA)n bases (occurring with delta H0 > 0 and delta C0P = 0) and (2) the binding of SSB to the unstacked DNA (occurring with delta H0 < 0 and delta C0P = 0). Therefore, although each isolated equilibrium occurs with delta C0P approximately 0, the overall equilibrium displays an apparent delta C0P,obs < 0 due to the coupled equilibrium. The binding of SSB to dT(pT)n and dC(pC)n occurs with delta H0 < 0 and delta C0P,obs = 0, since the bases in these ss-DNA molecules do not stack appreciably. These results indicate that a nonspecific protein-DNA interaction can display a large negative apparent delta C0P; however, this effect appears not to be due to the hydrophobic effect, but rather to a temperature-dependent conformational transition in the DNA that is coupled to protein binding. Implications of these observations for other protein-nucleic acid systems are discussed.

DNA↗
PubMed1994

Effects of base composition on the negative cooperativity and binding mode transitions of Escherichia coli SSB-single-stranded DNA complexes.

We have examined the ability of the Escherichia coli single-stranded DNA binding protein (SSB) tetramer to form its different binding modes on poly(dC), poly(U), and poly(A) over a range of NaCl and NaF concentrations for comparison with previous studies with poly(dT). In reverse titrations with poly(U) and poly(A) at 25 degrees C, pH 8.1, SSB forms all four binding modes previously observed with poly(dT), namely, (SSB)35, (SSB)40, (SSB)56, and (SSB)65, where the subscript denotes the site size (i.e., the average number of nucleotides occluded per SSB tetramer). As with poly(dT), the low site size modes are favored at low monovalent salt concentration (< 10 mM), whereas increasing salt concentration facilitates the transitions to the higher site size modes. Surprisingly, SSB does not form a stable (SSB)35 complex on poly(dC), even at 1 mM NaCl; rather, the (SSB)56 mode is formed under these conditions. Upon raising the [NaCl], the (SSB)56 complex undergoes a transition to the (SSB)65 complex (transition midpoint, 40 mM NaCl). On the basis of studies with dC(pC)34, dT(pT)34, and dA(pA)34, the inability of the SSB tetramer to form the (SSB)35 complex with poly(dC) is due mainly to a much lower degree of negative cooperativity for binding oligodeoxycytidylates to the SSB tetramer. At low salt concentration, the negative cooperativity parameter, sigma 35, is lowest for dA(pA)34, intermediate for dT(pT)34, and highest for dC(pC)34, indicating that it is most difficult to saturate the SSB tetramer with two molecules of dA(pA)34. We have also measured the equilibrium constants for binding the oligodeoxynucleotides dC(pC)34, dC(pC)69, dA(pA)34, and dA(pA)69 as a function of [NaCl] and [NaBr] and find that the salt dependencies of these oligonucleotides are dependent upon base composition. These studies also indicate that ion binding accompanies formation of these SSB-ss-DNA complexes, although there is a net release of ions upon formation of the complex. This influence of both salt concentration and base composition indicates that both electrostatic and nonelectrostatic factors contribute to the negative cooperativity associated with ss-DNA binding to the SSB tetramer.

Base Composition↗
PubMed1994

Co-operative binding of Escherichia coli SSB tetramers to single-stranded DNA in the (SSB)35 binding mode.

Escherichia coli SSB tetramers can bind to single stranded (ss) DNA in several binding modes. At 25 degrees C, pH 8.1, SSB can form at least three distinct binding modes, (SSB)n, where the number of nucleotides occluded per tetramer (n), can have values of 35, 56 or 65. Stability of the different modes is modulated by solution conditions, primarily the salt concentration and type, as well as the free SSB concentration. At least two different types of positive co-operative binding of SSB to ssDNA have also been observed, which appear to be correlated with different SSB binding modes. The (SSB)65 mode, which dominates at monovalent salt concentrations > 0.2 M, displays only moderate, "limited" co-operative binding in which clustering of SSB is limited to the formation of dimers of tetramers (octamers). However, at lower salt concentrations, "unlimited" co-operative binding is observed in which long SSB clusters can form, similar to the behavior observed for the phage T4 gene 32 protein. It has been proposed that unlimited co-operativity is linked to the (SSB)35 binding mode; however, this has not been verified since quantitative estimates of the co-operativity in this binding mode are difficult on long ssDNA. To estimate the nearest-neighbor co-operativity parameter in the (SSB)35 mode, we have examined the equilibrium binding of SSB to the oligodeoxynucleotide, dA(pA)69. Under certain conditions, 1:1 complexes, in which all four SSB subunits interact with the dA(pA)69, can form at low SSB binding densities, whereas 2:1 complexes, in which both SSB tetramers bind to DNA using only two subunits, can form at high SSB binding densities. These 2:1 complexes serve as a model for co-operative binding in the (SSB)35 binding mode. We show that SSB tetramers bind in this mode with a minimum nearest-neighbor co-operativity parameter of omega 35 = 1.0 x 10(5) (0.125 M NaCl, pH 8.1, 25 degrees C). This indicates that the nearest-neighbor co-operativities for SSB tetramers bound to ssDNA in the (SSB)35 versus the (SSB)65 mode differ qualitatively and quantitatively and suggests that the (SSB)35 mode is responsible for the ability of SSB protein to form long clusters on ssDNA. If the ability of helix destabilizing proteins to form uninterrupted protein clusters on ssDNA is important in DNA replication, then it is likely that SSB uses its (SSB)35 mode to function in this capacity.

Binding Sites↗
PubMed1994

Linkage of pH, anion and cation effects in protein-nucleic acid equilibria. Escherichia coli SSB protein-single stranded nucleic acid interactions.

We have examined the linkage between pH and monovalent salt concentration (NaCl and NaF) on the equilibrium binding of the Escherichia coli SSB protein to single stranded poly(U) in its (SSB)65 binding mode. In this mode, single-stranded nucleic acid interacts with all four subunits of the SSB tetramer covering approximately 65 nucleotides and nearest-neighbor cooperative interactions can form between DNA bound SSB tetramers, although protein clusters are limited to dimers of tetramers (octamers). The intrinsic association equilibrium constant, K(obs), and the "limited" cooperativity parameter, omega T/O, have been determined from titrations that monitor the quenching of the SSB tryptophan fluorescence upon binding poly(U). The cooperativity parameter, omega T/O, is independent of salt concentration and type and increases only slightly with increasing pH. However, K(obs) decreases with increasing salt concentration due to a net release of ions accompanying complex formation. This net ion release has contributions from cation release from the nucleic acid as well as differential binding of both cations and anions to the protein. The dependence of K(obs) on [NaF] is independent of pH with delta logK(obs)/delta log[NaF] = -4.5(+/- 0.5). However, there is a strong linkage between the effects of [NaCl] and pH, such that (delta logK(obs)/delta log[NaCl]) ranges from -12.0(+/- 0.8) at pH 5.5, to -6.0(+/- 0.5) at pH 9.0 (at 25 degrees C). Thus Cl- release increases with decreasing pH due to a linkage between chloride binding and protonation of the protein, whereas there is essentially no release of F-. The linkages of ion concentration and pH on K(obs) can be described in terms of: (1) cation release from the polynucleotide; (2) release of Cl- from sites on the SSB tetramer that require protonation to bind Cl-; (3) binding of cations to sites on the SSB tetramer which require deprotonation for cation binding, and (4) required binding of two-to-three protons by the SSB tetramer in order to form the SSB-poly(U) complex. Thus, the influence of salt concentration on protein-nucleic acid equilibria can be quite complex with contributions from differential ion binding to both the protein and the nucleic acid; however, these can be resolved by examining the linked effects of pH and salt concentration on these interactions.

Anions↗
PubMed1994

Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities.

There are now several well-documented SSBs from both prokaryotes and eukaryotes that function in replication, recombination, and repair; however, no "consensus" view of their interactions with ssDNA has emerged. Although these proteins all bind preferentially and with high affinity to ssDNA, their modes of binding to ssDNA in vitro, including whether they bind with cooperativity, often differ dramatically. This point is most clear upon comparing the properties of the phage T4 gene 32 protein and the E. coli SSB protein. Depending on the solution conditions, Eco SSB can bind ssDNA in several different modes, which display quite different properties, including cooperativity. The wide range of interactions with ssDNA observed for Eco SSB is due principally to its tetrameric structure and the fact that each SSB protomer (subunit) can bind ssDNA. This reflects a major difference between Eco SSB and the T4 gene 32 protein, which binds DNA as a monomer and displays "unlimited" positive cooperativity in its binding to ssDNA. The Eco SSB tetramer can bind ssDNA with at least two different types of nearest-neighbor positive cooperativity ("limited" and "unlimited"), as well as negative cooperativity among the subunits within an individual tetramer. In fact, this latter property, which is dependent upon salt concentration and nucleotide base composition, is a major factor influencing whether ssDNA interacts with all four or only two SSB subunits, which in turn determines the type of intertetramer positive cooperativity. Hence, it is clear that the interactions of Eco SSB with ssDNA are quite different from those of T4 gene 32 protein, and the idea that all SSBs bind to ssDNA as does the T4 gene 32 protein must be amended. Although it is not yet known which of the Eco SSB-binding modes is functionally important in vivo, it is possible that some of the modes are used preferentially in different DNA metabolic processes. In any event, the vastly different properties of the Eco SSB-binding modes must be considered in studies of DNA replication, recombination, and repair in vitro. Since eukaryotic mitochondrial SSBs as well as SSBs encoded by prokaryotic conjugative plasmids are highly similar to Eco SSB, these proteins are likely to show similar complexities. However, based on their heterotrimeric subunit composition, the eukaryotic nuclear SSBs (RP-A proteins) are significantly different from either Eco SSB or T4 gene 32 proteins. Further subclassification of these proteins must await more detailed biochemical and biophysical studies.

Allosteric Regulation↗
PubMed1993

Thermodynamics of single-stranded RNA and DNA interactions with oligolysines containing tryptophan. Effects of base composition.

We have examined the thermodynamics of binding of a series of oligolysines (net charge z = +2 to +10) containing one, two, or three tryptophans to several single-stranded (ss) homo-polynucleotides [poly(A), poly(C), poly(I), poly(dU), poly(dT)] and duplex (ds) DNA in order to investigate the effects of peptide charge, tryptophan content, and polynucleotide base and sugar type. Equilibrium association constants, Kobs, were measured as a function of monovalent salt concentration (KCH3CO2) and temperature by monitoring the quenching of the peptide tryptophan fluorescence upon interaction with the polynucleotides, from which the dependence of delta G(o)obs, delta H(o)obs, and delta S(o)obs on [KCH3CO2] was obtained. As observed previously with poly(U) [Mascotti, D.P., & Lohman, T.M. (1992) Biochemistry 31, 8932], the dependence of delta G(o)obs on [K+] for peptide binding to each polynucleotide is entirely entropic in origin (i.e., delta H(o)obs is independent of [K+]), consistent with the conclusion that Kobs increases with decreasing salt concentration due to the favorable increase in entropy resulting from the displacement of bound cations (K+) from the nucleic acid upon formation of the complex. For each ss polynucleotide, we find that significantly less than one potassium ion is released thermodynamically per net positive peptide charge, as determined from the value of delta log Kobs/delta log [K+]. Interestingly, (-delta log Kobs/delta log [K+])/z decreases with increasing peptide charge for poly(A), poly(C), and poly(dT), contrary to the behavior observed with poly(U) and ds-DNA, which may reflect a significant release of bound water upon formation of peptide complexes with these ss homo-polynucleotides or an increased binding of K+ to the ss polynucleotide with increasing [K+]. Alternatively, there may be conformational differences between the bound states of oligolysines of low charge, relative to oligolysines of higher charge. However, in all cases, peptides with z < +4 display different thermodynamics of binding than peptides with z > +4. The presence of tryptophan (Trp) within these peptides does not influence the salt dependence of Kobs for binding to poly(A), poly(C), or poly(dT). However, the Trp content of the peptide does contribute significantly to the thermodynamics of these interactions: Trp interactions result in a favorable contribution to delta H(o)obs, but an unfavorable contribution to delta S(o)obs, with little effect on delta G(o)obs due to entropy-enthalpy compensations. Oligolysines containing Trp also display a small, but significant, dependence of Kobs on base composition, with Kobs decreasing in the order poly(I) >> poly(dT) approximately poly(U) approximately poly(A) >> poly(C).

Amino Acid Sequence↗