Kinetic mechanism of direct transfer of Escherichia coli SSB tetramers between single-stranded DNA molecules

The kinetic mechanism of transfer of the homotetrameric Escherichia coli SSB protein between ssDNA molecules was studied using stopped-flow experiments. Dissociation of SSB from the donor ssDNA was monitored after addition of a large excess of unlabeled acceptor ssDNA by using either SSB tryptophan fluorescence or the fluorescence of a ssDNA labeled with an extrinsic fluorophore [fluorescein (F) or Cy3]. The dominant pathway for SSB dissociation occurs by a "direct transfer" mechanism in which an intermediate composed of two DNA molecules bound to one SSB tetramer forms transiently prior to the release of the acceptor DNA. When an initial 1:1 SSB-ssDNA complex is formed with (dT)(70) in the fully wrapped (SSB)(65) mode so that all four SSB subunits are bound to (dT)(70), the formation of the ternary intermediate complex occurs slowly with an apparent bimolecular rate constant, k(2,app), ranging from 1.2 x 10(3) M(-1) s(-1) (0.2 M NaCl) to approximately 5.1 x 10(3) M(-1) s(-1) (0.4 M NaBr), and this rate limits the overall rate of the transfer reaction (pH 8.1, 25 degrees C). These rate constants are approximately 7 x 10(5)- and approximately 7 x 10(4)-fold lower, respectively, than those measured for binding of the same ssDNA to an unligated SSB tetramer to form a singly ligated complex. However, when an initial SSB-ssDNA complex is formed with (dT)(35) so that only two SSB subunits interact with the DNA in an (SSB)(35) complex, the formation of the ternary intermediate occurs much faster with a k(2,app) ranging from >6.3 x 10(7) M(-1) s(-1) (0.2 M NaCl) to 2.6 x 10(7) M(-1) s(-1) (0.4 M NaBr). For these experiments, the rate of dissociation of the donor ssDNA determines the overall rate of the transfer reaction. Hence, an SSB tetramer can be transferred from one ssDNA molecule to another without proceeding through a free protein intermediate, and the rate of transfer is determined by the availability of free DNA binding sites within the initial SSB-ssDNA donor complex. Such a mechanism may be used to recycle SSB tetramers between old and newly formed ssDNA regions during lagging strand DNA replication.     

Microsecond dynamics of protein-DNA interactions: direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer

The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)(65) mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)(65) complex formation using the oligodeoxynucleotide, (dT)70, were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the approximately 500 ns to 4 ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)(65) complex.     

Plasmodium falciparum SSB tetramer binds single-stranded DNA only in a fully wrapped mode

The tetrameric Escherichia coli single-stranded DNA (ssDNA) binding protein (Ec-SSB) functions in DNA metabolism by binding to ssDNA and interacting directly with numerous DNA repair and replication proteins. Ec-SSB tetramers can bind ssDNA in multiple DNA binding modes that differ in the extent of ssDNA wrapping. Here, we show that the structurally similar SSB protein from the malarial parasite Plasmodium falciparum (Pf-SSB) also binds tightly to ssDNA but does not display the same number of ssDNA binding modes as Ec-SSB, binding ssDNA exclusively in fully wrapped complexes with site sizes of 52-65 nt/tetramer. Pf-SSB does not transition to the more cooperative (SSB)(35) DNA binding mode observed for Ec-SSB. Consistent with this, Pf-SSB tetramers also do not display the dramatic intra-tetramer negative cooperativity for binding of a second (dT)(35) molecule that is evident in Ec-SSB. These findings highlight variations in the DNA binding properties of these two highly conserved homotetrameric SSB proteins, and these differences might be tailored to suit their specific functions in the cell.     

Plasmodium falciparum SSB tetramer wraps single-stranded DNA with similar topology but opposite polarity to E. coli SSB

Single-stranded DNA binding (SSB) proteins play central roles in genome maintenance in all organisms. Plasmodium falciparum, the causative agent of malaria, encodes an SSB protein that localizes to the apicoplast and likely functions in the replication and maintenance of its genome. P. falciparum SSB (Pf-SSB) shares a high degree of sequence homology with bacterial SSB proteins but differs in the composition of its C-terminus, which interacts with more than a dozen other proteins in Escherichia coli SSB (Ec-SSB). Using sedimentation methods, we show that Pf-SSB forms a stable homo-tetramer alone and when bound to single-stranded DNA (ssDNA). We also present a crystal structure at 2.1 ? resolution of the Pf-SSB tetramer bound to two (dT)(35) molecules. The Pf-SSB tetramer is structurally similar to the Ec-SSB tetramer, and ssDNA wraps completely around the tetramer with a "baseball seam" topology that is similar to Ec-SSB in its "65 binding mode". However, the polarity of the ssDNA wrapping around Pf-SSB is opposite to that observed for Ec-SSB. The interactions between the bases in the DNA and the amino acid side chains also differ from those observed in the Ec-SSB-DNA structure, suggesting that other differences may exist in the DNA binding properties of these structurally similar proteins.     

Multiprotein E. coli SSB–ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.     

SSB蛋白与ssDNA相互作用的动力学和可视化研究

研究大肠杆菌单链结合蛋白(single-stranded DNA-binding protein,SSB)与单链DNA(single-stranded DNA,ssDNA)的相互作用对于了解其在DNA复制、重组和修复中的作用是非常重要的。通过表面等离子共振技术(surface plasmon resonance,SPR)得到了在有、无镁离子的情况下,SSB与ssDNA两者的平衡解离常数(equilibrium dissociation constant,KD)分别为9.67×10-7M和4.79×10-7M,阐明了镁离子对于两者作用形式的影响。利用原子力显微镜技术分别观察SSB蛋白、ssDNA和SSB-ssDNA复合物的成像,为下一步研究SSB在DNA代谢中作用模式的单分子可视化奠定了基础。     

Probing E. coli SSB protein-DNA topology by reversing DNA backbone polarity

Escherichia coli single-strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in two major modes, differing in occluded site size and cooperativity. The (SSB)₃₅ mode in which ssDNA wraps, on average, around two subunits is favored at low [NaCl] and high SSB/DNA ratios and displays high unlimited, nearest-neighbor cooperativity forming long protein clusters. The (SSB)₆₅ mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (>200 mM) and displays limited low cooperativity. Crystal structures of E. coli SSB and Plasmodium falciparum SSB show ssDNA bound to the SSB subunits (OB folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)₇₀ constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse-polarity (RP) phosphodiester linkage, either 3′-3′ or 5′-5′. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)₇₀ with a 3′-3′ polarity switch shows decreased affinity for EcSSB in the (SSB)₆₅ mode and lower cooperativity in the (SSB)₃₅ mode. However, (dT)₇₀ in which every phosphodiester linkage is reversed does not form a completely wrapped (SSB)₆₅ mode but, rather, binds EcSSB in the (SSB)₃₅ mode with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)₆₅ mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB folds if there is a change in ssDNA wrapping topology of the RP ssDNA.     

Monomers of the Escherichia coli SSB-1 mutant protein bind single-stranded DNA

The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair. The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication. Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT)15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding. At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT)15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e. negative co-operativity is observed for ssDNA binding to the SSB-1 protomers. As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT)15 binding to the tetramer in each phase. However, the intrinsic binding constant, K16, for the SSB-1 protomer-dT(pT)15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, sigma 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity. At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT)15 without negative co-operativity; however, the intrinsic affinity of dT(pT)15 for the monomer is a factor of approximately 10 lower than for the protomer (50 mM-NaCl, pH 8.1, 25 degrees C). Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly. These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer. On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.     

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)     

Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer binding to single-stranded DNA

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (DeltaH(obs)) and the negative heat capacity change (DeltaC(p,obs)) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined DeltaH(obs) over a much wider temperature range (5-60 degrees C) and as a function of monovalent salt concentration and type for SSB binding to (dT)(70) under solution conditions that favor the fully wrapped (SSB)(65) complex (monovalent salt concentration >or=0.20 M). Over this wider temperature range we observe a strongly temperature-dependent DeltaC(p,obs). The DeltaH(obs) decreases as temperature increases from 5 to 35 degrees C (DeltaC(p,obs) <0) but then increases at higher temperatures up to 60 degrees C (DeltaC(p,obs) >0). Both salt concentration and anion type have large effects on DeltaH(obs) and DeltaC(p,obs). These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 degrees C), the midpoint of which shifts to higher temperature (above 35 degrees C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br(-) > Cl(-) > F(-), and these anions are then released upon binding ssDNA, affecting both DeltaH(obs) and DeltaC(p,obs). We conclude that the experimentally measured values of DeltaC(p,obs) for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.     

Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein

Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)(35) and (SSB)(65) formed on (dT)(70), with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)(35) mode, suggesting that interactions of proteins with the C terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.     

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.     

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.     

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.     

Single-stranded DNA binding protein from calf thymus. Purification, properties, and stimulation of the homologous DNA-polymerase-alpha-primase complex.

A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA-cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius approximately 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11-kDa complex. The ssDNA binding protein (SSB) covered approximately 20-25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-alpha-primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA).(dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA).(dT)14; with poly(dT).(rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase alpha to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300-600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentration-dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.     

Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes

We have examined the single-stranded DNA (ssDNA) binding properties of the Saccharomyces cerevisiae replication protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry, and sedimentation equilibrium to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants, and binding enthalpy of scRPA-(dT)L complexes as a function of the oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is a stable heterotrimer over the range of [NaCl] examined (0.02-1.5 M). However, the occluded site size, n, undergoes a salt-dependent transition between values of n = 18-20 nucleotides at low [NaCl] and values of n = 26-28 nucleotides at high [NaCl], with a transition midpoint near 0.36 M NaCl (25.0 degrees C, pH 8.1). Measurements of the stoichiometry of scRPA-(dT)L complexes also show a [NaCl]-dependent change in stoichiometry consistent with the observed change in the occluded site size. Measurements of the deltaH(obsd) for scRPA binding to (dT)L at 1.5 M NaCl yield a contact site size of 28 nucleotides, similar to the occluded site size determined at this [NaCl]. Altogether, these data support a model in which scRPA can bind to ssDNA in at least two binding modes, a low site size mode (n = 18 +/- 1 nucleotides), stabilized at low [NaCl], in which only three of its oligonucleotide/oligosaccharide binding folds (OB-folds) are used, and a higher site size mode (n = 27 +/- 1 nucleotides), stabilized at higher [NaCl], which uses four of its OB-folds. No evidence for highly cooperative binding of scRPA to ssDNA was found under any conditions examined. Thus, scRPA shows some behavior similar to that of the E. coli SSB homotetramer, which also shows binding mode transitions, but some significant differences also exist.     

E. coli SSB tetramer binds the first and second molecules of (dT)(35) with heat capacities of opposite sign

We have previously shown that formation of a 1:1 fully wrapped complex of Escherichia coli SSB tetramer with (dT)₇₀ displays a temperature-dependent sign reversal of the binding heat capacity (ΔC_P). Here we examine SSB binding to shorter oligodeoxynucleotides ((dX)₃₅) to probe whether this effect requires binding of one or two (dX)₃₅ molecules per SSB tetramer. We find that the ΔC_P for the first molecule of (dX)₃₅ is always negative. However, a sign reversal of ΔC_P from negative to positive occurs with increasing temperature for binding of the second (dX)₃₅. This striking behavior of ΔC_P for the second (dX)₃₅ appears linked to conformational changes within the ssDNA-SSB complex that are required to form a fully wrapped (SSB)₆₅ binding mode. These results also underscore that binding heat capacities of macromolecular interactions have multiple origins that cannot be understood simply on the basis of examining static structures.     

Adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates.

Isothermal titration calorimetry (ITC) was used to test the hypothesis that the relatively small enthalpy change (DeltaHobs) and large negative heat capacity change (DeltaCp,obs) observed for the binding of the Escherichia coli SSB protein to single-stranded (ss) oligodeoxyadenylates result from the temperature-dependent adenine base unstacking equilibrium that is thermodynamically coupled to binding. We have determined DeltaH1,obs for the binding of 1 mole of each of dT(pT)34, dC(pC)34, and dA(pA)34 to the SSB tetramer (20 mM NaCl at pH 8.1). For dT(pT)34 and dC(pC)34, we found large, negative values for DeltaH1,obs of -75 +/- 1 and -85 +/- 2 kcal/mol at 25 degrees C, with DeltaCp,obs values of -540 +/- 20 and -570 +/- 30 cal mol-1 K-1 (7-50 degrees C), respectively. However, for SSB-dA(pA)34 binding, DeltaH1,obs is considerably less negative (-14 +/- 1 kcal/mol at 25 degrees C), even becoming positive at temperatures below 13 degrees C, and DeltaCp,obs is nearly twice as large in magnitude (-1180 +/- 40 cal mol-1 K-1). These very different thermodynamic properties for SSB-dA(pA)34 binding appear to result from the fact that the bases in dA(pA)34 are more stacked at any temperature than are the bases in dC(pC)34 or dT(pT)34 and that the bases become unstacked within the SSB-ssDNA complexes. Therefore, the DeltaCp,obs for SSB-ssDNA binding has multiple contributions, a major one being the coupling to binding of a temperature-dependent conformational change in the ssDNA, although SSB binding to unstacked ssDNA still has an "intrinsic" negative DeltaCp,0. In general, such temperature-dependent changes in the conformational "end states" of interacting macromolecules can contribute significantly to both DeltaCp,obs and DeltaHobs.     

Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration

The binding properties of the Escherichia coli encoded single strand binding protein (SSB) to a variety of synthetic homopolynucleotides, as well as to single stranded M13 DNA, have been examined as a function of the NaCl concentration (25.0 degrees C, pH 8.1). Quenching of the intrinsic tryptophan fluorescence of the SSB protein by the nucleic acid is used to monitor binding. We find that the site size (n) for binding of SSB to all single stranded nucleic acids is quite dependent on the NaCl concentration. For SSB-poly(dT), n = 33 +/- 3 nucleotides/tetramer below 10 mM NaCl and 65 +/- 5 nucleotides/tetramer above 0.20 M NaCl (up to 5 M). Between 10 mM and 0.2 M NaCl, the apparent site size increases continuously with [NaCl]. The extent of quenching of the bound SSB fluorescence by poly(dT) also displays two-state behavior, 51 +/- 3% quenching below 10 mM NaCl and 83 +/- 3% quenching at high [NaCl] (greater than 01.-0.2 M NaCl), which correlates with the observed changes in the occluded site size. On the basis of these observations as well as the data of Krauss et al. (Krauss, G., Sindermann, H., Schomburg, U., and Maass, G. (1981) Biochemistry 20, 5346-5352) and Chrysogelos and Griffith (Chrysogelos, S., and Griffith, J. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,5803-5807) we propose a model in which E. coli SSB binds to single stranded nucleic acids in two binding modes, a low salt mode (n = 33 +/- 3), referred to as (SSB)33, in which the nucleic acid interacts with only two protomers of the tetramer, and one at higher [NaCl], n = 65 +/- 5, (SSB)65, in which the nucleic acid interacts with all 4 protomers of the tetramer. At intermediate NaCl concentrations a mixture of these two binding modes exists which explains the variable site sizes and other apparent discrepancies previously reported for SSB binding. The transition between the two binding modes is reversible, although the kinetics are slow, and it is modulated by NaCl concentrations within the physiological range. We suggest that SSB may utilize both binding modes in its range of functions (replication, recombination, repair) and that in vivo changes in the ionic media may play a role in regulating some of these processes.