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.     

Stopped-flow studies of the kinetics of single-stranded DNA binding and wrapping around the Escherichia coli SSB tetramer

We have examined the kinetic mechanism for binding of the homotetrameric Escherichia coliSSB protein to single-stranded oligodeoxynucleotides [(dT)(70) and (dT)(35)] under conditions that favor the formation of a fully wrapped ssDNA complex in which all four subunits interact with DNA. Under these conditions, a so-called (SSB)(65) complex is formed in which either one molecule of (dT)(70) or two molecules of (dT)(35) bind per tetramer. Stopped-flow studies monitoring quenching of the intrinsic SSB Trp fluorescence were used to examine the initial binding step. To examine the kinetics of ssDNA wrapping, we used a single-stranded oligodeoxythymidylate, (dT)(66), that was labeled on its 3'-end with a fluorescent donor (Cy3) and on its 5'-end with a fluorescent acceptor (Cy5). Formation of the fully wrapped structure was accompanied by extensive fluorescence resonance energy transfer (FRET) from Cy3 to Cy5 since the two ends of (dT)(66) are in close proximity in the fully wrapped complex. Our results indicate that initial ssDNA binding to the tetramer is very rapid, with a bimolecular rate constant, k(1,app), of nearly 10(9) M(-1) s(-1) in the limit of low salt concentration (<0.2 M NaCl, pH 8.1, 25.0 degrees C), whereas the rate of dissociation is very low at all salt concentrations that were examined (20 mM to 2 M NaCl or NaBr). However, the rate of initial binding and the rate of formation of the fully wrapped complex are identical, indicating that the rate of wrapping of the ssDNA around the SSB tetramer is very rapid, with a lower limit rate of 700 s(-1). The implications of this rapid binding and wrapping reaction are discussed.     

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.     

Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein: Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 °C) and magnesium chloride concentrations of 1 mm or 4 mm, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 °C) and magnesium chloride concentrations of 4 mm or 10 mm, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(±1) nucleotides per RecA protein monomer at 4 mm-MgCl₂ and 37 °C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mm-MgCl₂. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA. In addition, our results have disclosed an additional property of the RecA protein-single-stranded DNA complex: namely, in the presence of complementary base-pairing and at elevated temperatures and magnesium concentrations, a unique RecA protein-DNA complex forms that is resistant to inhibition by SSB protein.     

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

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

Mechanism of stimulation of T7 DNA polymerase by Escherichia coli single-stranded DNA binding protein (SSB)

Single-stranded DNA binding protein is a key component in growth of bacteriophage T7. In addition, DNA synthesis by the purified in vitro replication system is markedly stimulated when the DNA template is coated with Escherichia coli single-stranded DNA binding protein (SSB). In an attempt to understand the mechanism for this stimulation, we have studied the effect of E. coli SSB on DNA synthesis by the T7 DNA polymerase using a primed single-stranded M13 DNA template which serves as a model for T7 lagging strand DNA synthesis. Polyacrylamide gel analysis of the DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase pauses at many specific sites, some stronger than others. By comparing the position of pausing with the DNA sequence of this region and by using a DNA template that contains an extremely stable hairpin structure, it was found that many, but not all, of these pause positions correspond to regions of potential secondary structure. The presence of SSB during synthesis resulted in a large reduction in the frequency of hesitations at many sites that correspond to these secondary structures. However, the facts that a large percentage of the pause sites remain unaffected even at saturating levels of SSB and that SSB stimulates synthesis on a singly primed poly(dA) template suggested that other mechanisms also contribute to the stimulation of DNA synthesis caused by SSB. Using a sucrose gradient analysis, we found that SSB increases the affinity of the polymerase for single-stranded DNA that this increased binding is only noticed when the polymerase concentration is limiting. The effect of this difference in polymerase affinity was clearly observed by a polyacrylamide gel analysis of the product DNA synthesized during a limited DNA synthesis reaction using conditions where only two nucleotides are added to the primer. Under these circumstances, where the presence of hairpin structures should not contribute to the stimulatory effect of SSB, we found that the extension of the primer is stimulated 4-fold if the DNA template is coated with SSB. Furthermore, SSB had no effect on this synthesis at large polymerase to template ratios.     

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.     

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.     

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.     

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein

Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔH_obs, and accounts for as much as a half of the observed heat capacity change, ΔC_p. However, there is still a substantial ΔC_p associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)₃₄ to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)₃₄ binding over this entire pH range, with contributions from at least three sets of protonation sites (pK_a1 = 5.9–6.6, pK_a2 = 8.2–8.4, and pK_a3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔC_p for the SSB‐dT(pT)₃₄ interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol⁻¹ K⁻¹) accounts for as much as half of the total ΔC_p. The values of the “intrinsic” ΔC_p,0 range from −210 ± 33 cal mol⁻¹ °K⁻¹ to −237 ± 36 cal mol⁻¹K⁻¹, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔC_p, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc.     

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.     

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

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

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

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

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

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

A dimeric mutant of the homotetrameric single-stranded DNA binding protein from Escherichia coli

A single amino acid substitution (Y78R) at the dimer-dimer interface of homotetrameric single stranded DNA binding protein from E. coli (EcoSSB) renders the protein a stable dimer. This dimer can bind single-stranded DNA albeit with greatly reduced affinity. In vivo this dimeric SSB cannot replace homotetrameric EcoSSB. Amino acid changes at the rim of the dimer-dimer interface nearby (Q76K, Q76E) show an electrostatic interaction between a charged amino acid at position 76 and bound nucleic acid. In conclusion, nucleic acid binding to homotetrameric SSB must take place across both dimers to achieve functionally correct binding.     

Chimeras of Escherichia coli and Mycobacterium tuberculosis single-stranded DNA binding proteins: characterization and function in Escherichia coli

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mβ1, mβ1'β2, mβ1-β5, mβ1-β6 and mβ4-β5) by transplanting β1, β1'β2, β1-β5, β1-β6 and β4-β5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mβ1'β2(ESWR) SSB was generated by mutating the MtuSSB specific 'PRIY' sequence in the β2 strand of mβ1'β2 SSB to EcoSSB specific 'ESWR' sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mβ1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mβ1-β6, MtuSSB, mβ1'β2 and mβ1-β5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1'β2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.     

Studies on the noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded nucleic acids.

The noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA oligomers has been studied by means of equilibrium dialysis. Dialyses were performed under a number of solution and temperature conditions using oligomers of varying length and base compositions. The results of these studies, which include a Scatchard analysis of the binding, have allowed us to propose a model for the cooperative binding of the protein to single-stranded DNA. The results of experiments dealing with the interaction of the protein with single-stranded RNA are also presented.