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PubMed1982

Effects of the ssb-1 and ssb-113 mutations on survival and DNA repair in UV-irradiated delta uvrB strains of Escherichia coli K-12.

The molecular defect in DNA repair caused by ssb mutations (single-strand binding protein) was studied by analyzing DNA synthesis and DNA double-strand break production in UV-irradiated Escherichia coli delta uvrB strains. The presence of the ssb-113 mutation produced a large inhibition of DNA synthesis and led to the formation of double-strand breaks, whereas the ssb-1 mutation produced much less inhibition of DNA synthesis and fewer double-strand breaks. We suggest that the single-strand binding protein plays an important role in the replication of damaged DNA, and that it functions by protecting single-stranded parental DNa opposite daughter-strand gaps from nuclease attack.

Cell Survival↗

PubMed2005

The SPRY domain-containing SOCS box protein 1 (SSB-1) interacts with MET and enhances the hepatocyte growth factor-induced Erk-Elk-1-serum response element pathway.

The suppressor of cytokine signaling (SOCS) protein family includes a SPRY (repeats in splA and RyR) domain-containing SOCS box protein (SSB) subfamily, which consists of four members, SSB-1, SSB-2, SSB-3, and SSB-4. These proteins contain a central SPRY domain and a C-terminal SOCS box. Although some of the SOCS protein subfamilies function as adaptors for a large family of ubiquitin-protein isopeptide ligases to regulate certain signaling pathways, the function of the SSB subfamily remains to be determined. In our previous studies, we have found that two SPRY domain-containing proteins, RanBP9 and RanBP10, interact with MET through the SPRY domain. In the present study, we explored the function of SSB proteins in the regulation of the hepatocyte growth factor (HGF)-MET signaling. Our results showed that all four SSB proteins also interacted with the MET. The MET interaction with SSB-1 was further investigated. We demonstrated that SSB-1 bound to MET tyrosine kinase domain through its SPRY domain. MET interacted with SSB-1 in both the absence and the presence of HGF, but HGF treatment resulted in the recruitment of more SSB-1 by MET. We showed that overexpression of SSB-1 but not other SSB proteins enhanced the HGF-induced serum response element (SRE)-luciferase activity. Overexpression of SSB-1 exhibited no effect on the basal level or epidermal growth factor-induced SRE-luciferase activity. SSB-1 also enhanced HGF-induced Erk phosphorylation. Suppression of SSB-1 by the RNA interference method down-regulated HGF-induced SRE-luciferase activity and decreased Elk-1 activation. These results suggest that SSB-1 may play an important role in enhancing the HGF-induced Erk-Elk-1-SRE pathway. Furthermore, we demonstrated that in response to HGF stimulation, the SSB-1 protein became phosphorylated at tyrosine residue 31. The phosphorylated SSB-1 protein bound to p120Ras-GTPase-activating protein (GAP) but did not promote the degradation of p120RasGAP, indicating that enhanced HGF-MET signaling by overexpression of SSB-1 was not dependent on p120RasGAP degradation.

Amino Acid Sequence↗

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↗

PubMed1991

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.

Bacterial Proteins↗

PubMed2002

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.

Binding Sites↗

PubMed1984

Quantification of SSB protein in E. coli and its variation during RECA protein induction.

Using a two-site immunometric assay (IRMA) we quantified the concentration of single-stranded DNA binding protein (SSB) in several E. coli strains. We found approximately 7,000 monomers of SSB present per bacterium, and this number remained constant throughout the exponential phase of growth. Two ssb- mutants (ssb-1 and ssb-113) are defective in the induction of the S.O.S. pathway. One of the first functions expressed upon induction of the S.O.S. pathway is the amplification of recA protein (RECA), which we monitored by an IRMA assay similar to the one used for SSB quantification. By combining the two assays we determined the level of SSB and RECA in ssb- mutants or in SSB and RECA overproducer strains. We found: a) a normal induction of RECA following UV irradiation of E. coli bacteria overproducing SSB, b) a normal level of SSB in wild type and ssb-1 and ssb-113 mutants either in the absence or in the presence of S.O.S. inducing agents. We confirmed a severe impairment in the induction of RECA in these two mutants after nalidixic acid treatment. Our results suggest that the concentrations of RECA and SSB protein in E. coli are regulated by independent biochemical pathways.

DNA-Binding Proteins↗

PubMed1988

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

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

Bacterial Proteins↗

PubMed1985

A monoclonal antibody that recognizes the functional domain of Escherichia coli single-stranded DNA binding protein that includes the ssb-113 mutation.

We have isolated a monoclonal antibody against Escherichia coli single-stranded DNA binding protein (SSB) that recognizes the functional domain specified by the ssb-113 temperature-sensitive mutation, a domain which is distinct from the DNA-binding site. Although the ssb-113 and ssb-1 mutations result in many similar phenotypic defects, they differ significantly in others, indicating that they affect different functional domains of the protein. Whereas the SSB-1 mutant protein is clearly defective in tetramer formation and is also unable to bind single-stranded DNA at nonpermissive temperatures, no similar in vitro defects have yet been found in the SSB-113 mutant protein. In fact, the only reported in vitro effect of the ssb-113 mutation on the protein is a slight increase in its helix destabilizing ability. Competition radioimmunoassays using a monoclonal antibody demonstrated that SSB-113 mutant protein, containing a single amino acid substitution at position 176 (the penultimate residue), did not compete with SSB while SSB-1 protein (with a single change at position 55) did compete with SSB. This analysis was refined by studies with a proteolysis fragment and with peptides derived from both SSB and SSB-113. The results indicate that the antibody recognizes a determinant near the COOH-terminal end of the protein and that the SSB-113 mutation lies within or very close to this determinant.

Antibodies, Monoclonal↗

PubMed1987

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.

Adenosine Triphosphate↗

PubMed1989

The ssb gene of plasmid ColIb-P9.

The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12. The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein. Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region. High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA. A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth. Hence, no biological role of ColIb SSB protein was detected. However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins.

Amino Acid Sequence↗

PubMed1997

A mutation in E. coli SSB protein (W54S) alters intra-tetramer negative cooperativity and inter-tetramer positive cooperativity for single-stranded DNA binding.

E. coli SSB tetramer binds with high affinity and cooperatively to single-stranded (ss) DNA and functions in replication, recombination and repair. Curth et al. (Biochemistry, 32 (1993) 2585-2591) have shown that a mutant SSB protein, in which Trp-54 has been replaced by Ser (W54S) in each subunit, binds preferentially to ss-polynucleotides in the (SSB)35 mode in which only 35 nucleotides are occluded per tetramer under conditions in which wild-type (wt) SSB binds in its (SSB)65 mode. The W54S mutant also displays increased UV sensitivity and slow growth phenotypes, suggesting defects in vivo in both repair and replication (Carlini et al. (Molecular Microbiology, 10 (1993) 1067)). We have characterized the energetics of SSBW54S binding to poly(dT) as well as short oligodeoxyribonucleotides (dA(pA)69, dT(pT)34, dC(pC)34) to determine the basis for this dramatic change in binding mode preference. We find that the W54S mutant remains a stable tetramer; however, its affinity for ss-DNA as well as both the intra-tetramer negative cooperativity and its inter-tetramer positive cooperativity in the (SSB)35 mode (omega 35) are altered significantly compared to wtSSB. The increased intra-tetramer negative cooperativity makes it more difficult for ss-DNA to bind the third and fourth subunits of the W54S tetramer, explaining the increased stability of the (SSB)35 mode in complexes with poly(dT). When bound to dA(pA)69 in the (SSB)35 mode, W54S tetramer also displays a dramatically lower inter-tetramer positive cooperativity (omega 35 = 77(+/-20)) than wtSSB (omega 35 > or = 10(5)) as well as a significantly lower affinity for ss-DNA. These results indicate that a single amino acid change can dramatically influence the ability of SSB tetramers to bind in the different SSB binding modes. The altered ss-DNA properties of the W54S SSB mutant are probably responsible for the observed defects in replication and repair and support the proposal that the different SSB binding modes may function selectively in replication, recombination and/or repair.

Biopolymers↗

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↗

PubMed2001

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein.

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

Amino Acid Sequence↗

PubMed1984

Characterization of the Escherichia coli SSB-113 mutant single-stranded DNA-binding protein. Cloning of the gene, DNA and protein sequence analysis, high pressure liquid chromatography peptide mapping, and DNA-binding studies.

The ssb-113 (formerly lexC113) gene encoding a mutant single-stranded DNA binding protein (SSB) has been cloned into plasmid pSC101 resulting in 5- to 10-fold more mutant protein than strains carrying only one (chromosomal) copy of the gene. Analysis of tryptic and chymotryptic peptides of the mutant protein by high pressure liquid chromatography and solid phase protein sequencing has shown that the ssb-113 mutation results in the substitution of serine for proline at residue 176 of SSB. This change could only occur in one step by a C leads to T transition in the DNA sequence. Physicochemical studies of the homogeneous mutant protein have shown that it binds as well as wild type SSB to single-stranded DNA and that it is a slightly better helix-destabilizing protein than wild type SSB as measured by its ability to lower the thermal melting transition of poly[d(A-T)]. In vivo studies of ssb-113 strains carrying the cloned ssb-113 gene in pSC101 have shown that overproduction of the mutant protein does not complement the temperature-sensitive conditional lethality caused by the ssb-113 mutation when present in single gene copy in contrast to effects recently observed in ssb-1 strains overproducing the ssb-1 encoded protein (Chase, J. W., Murphy, J. B., Whittier, R. F., Lorensen, E., and Sninsky, J. J. (1983) J. Mol. Biol. 164, 193-211). Also noted in this report are two corrections to the DNA sequence of wild type SSB, one of which places glycine (codon GGC) at residue 133 rather than serine as previously reported (Sancar, A., Williams, K. R., Chase, J. W., and Rupp, W. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4274-4278). The second correction to the DNA sequence is in the serine 39 codon, previously reported to be TCA and now correctly shown to be TCC.

Amino Acid Sequence↗

PubMed1987

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

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

Allosteric Regulation↗

PubMed1988

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

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

Anions↗

PubMed1994

The single-stranded-DNA-binding proteins (SSB) of Proteus mirabilis and Serratia marcescens.

The single-stranded-DNA-binding (SSB) proteins from Proteus mirabilis and Serratia marcescens were purified from overproducing Escherichia coli strains, which were devoid of their own ssb gene. The strains harboured an endA insertion mutation and a xonA mutation resulting in the absence of endonuclease I and exonuclease I activities from the preparations. The amino acid sequences of the SSB of all three species are nearly identical in the N-terminal parts of the proteins that contain the DNA-binding domain, but differ in the C-terminal parts. Both proteins have an apparent binding-site size of 65 and 35 nucleotides at high and low salt concentrations, respectively. The association-rate constant for binding to poly(dT) is 3.2 x 10(8) M-1 s-1 for P. mirabilis SSB (PmiSSB) and 3.4 x 10(8) M-1 s-1 for S. marcescens SSB (SmaSSB). These binding parameters are very similar to those of E. coli SSB (EcoSSB). The structural similarity of the proteins is also documented by the finding that they can exchange subunits among each other to form mixed tetramers. The transcriptional regulation of the ssb and uvrA genes from P. mirabilis and S. marcescens in SOS-induced E. coli cells was studied using lacZ fusions. While the uvrA genes were inducible, there was no induction of the ssb genes transcribed divergently from the uvrA genes. Apparently, regions with nucleotide sequence similarity to the E. coli SOS-box preceding the ssb genes of P. mirabilis and S. marcescens had no gross effect on the transcription. Studies on growth of the cells and recovery from ultraviolet damage indicate that the heterologous SSB proteins support DNA replication and recombinational DNA repair of E. coli with the same efficiency as the E. coli SSB protein. Interactions with other E. coli proteins involved in these processes either do not occur, or are not impeded.

Amino Acid Sequence↗

PubMed1988

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.

Base Sequence↗