Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein

The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K. R., Murphy, J. B., and Chase, J. W. (1984) J. Biol. Chem. 259, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3. The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.     

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.     

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.     

Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene: the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage λ⁺, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA^? mutant strains and will provide a means of transferring ssb^? mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.     

Suppression of the ssb-1 and ssb-113 mutations of Escherichia coli by a wild-type rep gene, NaCl, and glucose

The ssb-1 mutation confers severe temperature sensitivity and UV sensitivity on many strains of Escherichia coli K-12 and C, including strain C1412. However, ssb-1 confers only slight temperature sensitivity and slight UV sensitivity on strain C1a, suggesting that strain C1a contains extragenic suppressors of ssb-1. We found that introduction of the wild-type rep gene from C1a into strain C1412 ssb-1 gave strong suppression of temperature sensitivity and moderate suppression of UV sensitivity. Also, the C1a rep+ gene mildly suppressed the temperature sensitivity conferred by the ssb-113 mutation, formerly called lexC113. Suppression of the C1412 ssb-1 growth defect by C1a rep+ rendered the cells Gro- for phi X174. In contrast to the positive suppression of ssb-1 and ssb-113 by a wild-type rep gene, mutant rep alleles enhanced the severity of the ssb-1 defect, with several C1a ssb-1 double mutants being either more temperature sensitive or more UV sensitive than C1a ssb-1, depending on which mutant rep allele was used. As a control, the same rep alleles in combination with a dnaB mutation gave an allele-independent increase in temperature sensitivity. Our results on suppression of ssb-1 by rep and on the role of the genetic background in this suppression suggested that the rep and ssb proteins interact to form a subcomplex of the total DNA replication complex and that this subcomplex has some function in repair. The effects of NaCl and glucose on suppression of both the temperature sensitivity and the UV sensitivity conferred by ssb-1 and ssb-113 are described. The degree of suppression of temperature sensitivity by salt or glucose was dependent on the source of the wild-type rep allele, as well as on the genetic background.     

Effects of various single-stranded-DNA-binding proteins on reactions promoted by RecA protein

To relate the roles of Escherichia coli SSB in recombination in vivo and in vitro, we have studied the mutant proteins SSB-1 and SSB-113, the variant SSBc produced by chymotryptic cleavage, the partially homologous variant F SSB (encoded by the E. coli sex factor), and the protein encoded by gene 32 of bacteriophage T4. All of these, with the exception of SSB-1, augmented both the initial rate of homologous pairing and strand exchange promoted by RecA protein. From these and related observations, we conclude that SSB stimulates the initial formation of joint molecules by nonspecifically promoting the binding of RecA protein to single-stranded DNA; that SSB plays no role in synapsis of the RecA nucleoprotein filament with duplex DNA; that stimulation of strand exchange by SSB is similarly nonspecific; and that all members of the class of proteins represented by SSB, F SSB, and gene 32 protein may play equivalent roles in making single-stranded DNA more accessible to RecA protein.     

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.     

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.     

Single-stranded DNA-binding protein recruits DNA polymerase V to primer termini on RecA-coated DNA

Translesion DNA synthesis (TLS) by DNA polymerase V (polV) in Escherichia coli involves accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in vitro exonuclease protection assay and found that SSB increases the accessibility of 3' primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defective in protein-protein interactions, but not in DNA binding, was as effective as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS. Consistently, the heterologous SSB proteins gp32, encoded by phage T4, and ICP8, encoded by herpes simplex virus 1, could replace E. coli SSB in the TLS reaction, albeit with lower efficiency. Immunoprecipitation experiments indicated that polV directly interacts with SSB and that this interaction is disrupted by the SSB-113 mutation. Taken together our results suggest that SSB functions to recruit polV to primer termini on RecA-coated DNA, operating by two mechanisms: 1) increasing the accessibility of 3' primer termini caused by binding of SSB to DNA and 2) a direct SSB-polV interaction mediated by the C terminus of SSB.     

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.     

Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46.

Previous studies from our laboratory have shown that an allele of the heat shock protein GroEL (groEL411) is able to specifically suppress some of the physiological defects of the single-stranded DNA-binding protein mutation ssb-1. A search for additional alleles of the groE genes which may act as suppressors for ssb mutations has led to the identification of groEL46 as a specific suppressor of ssb-113. It has very little or no effect on ssb-1 or ssb-3. All of the physiological defects of ssb-113, including temperature-sensitive growth, temperature-sensitive DNA synthesis, sensitivity to UV irradiation, methyl methanesulfonate, and bleomycin, and reduced recombinational capacity, are restored to wild-type levels. The ssb-113 allele, however, is unable to restore sensitivity of groEL46 cells to phage lambda. The mechanism of suppression of ssb-113 by groEL46 appears to differ from that of ssb-1 by groEL411. The data suggest that GroEL may interact with single-stranded DNA-binding protein in more than one domain.     

Interaction of E. coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease

The 3'-5' single-stranded DNA(ssDNA) degrading exonuclease I of E. coli directly interacts with the E. coli ssDNA binding protein (EcoSSB). Analytical ultracentrifugation shows that all 4 carboxy-termini of an EcoSSB tetramer bind exonuclease I. Binding is weakened by increasing salt concentrations, indicating the involvement of the negatively charged amino acids of the carboxy-terminus of SSB. Mutant SSB proteins EcoSSBP176S (ssb-113) and EcoSSBF177C do not bindtoexonuclease I while EcoSSBG15D (ssb-3) does bind. In a co-precipitation assay we show that the absence of the lastten amino acids (PMDFDDDIPF) completely abolishes binding of EcoSSB to exonuclease I. The interaction does not depend on the presence of the correct amino-terminal DNA binding domain or the amino acid sequences between the DNA binding domain and the last ten amino acids. A synthetic peptide (WMDFDDDIPF), corresponding to the last nine amino acids of EcoSSB, specifically inhibits the interaction. Both EcoSSBP176S and EcoSSBF177C SSBs bind DNA similar to wild-type EcoSSB, indicating that the phenotype of ssb-113 is not an indication of altered DNA binding. The repair deficiency of either ssb-3 or ssb-113 strain can be complemented by overexpression of the respective other mutant.     

Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli

Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein to tyrosine or lysine leads to cells which are UV- and temperature-sensitive. The defects of both ssbH55Y (ssb-1) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation producing a less stable, readily dissociating protein whose more severe replication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was suppressed by spontaneous mutations that changed the glutamine at position 76 or 110 to leucine. Using guanidinium chloride denaturation monitored by sedimentation diffusion equilibrium experiments in the analytical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable than wild-type SSB. Additionally, the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denaturation than wild-type SSB protein.     

The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase.

The glc7 mutant of the yeast Saccharomyces cerevisiae does not accumulate glycogen due to a defect in glycogen synthase activation (Peng, Z., Trumbly, R. J., and Reimann, E.M. (1990) J. Biol. Chem. 265, 13871-13877) whereas wild-type strains accumulate glycogen as the cell cultures approach stationary phase. We isolated the GLC7 gene by complementation of the defect in glycogen accumulation and found that the GLC7 gene is the same as the DIS2S1 gene (Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007). The protein product predicted by the GLC7 DNA sequence has a sequence that is 81% identical with rabbit protein phosphatase 1 catalytic subunit. Protein phosphatase 1 activity was greatly diminished in extracts from glc7 mutant cells. Two forms of protein phosphatase 1 were identified after chromatography of extracts on DEAE-cellulose. Both forms were diminished in the glc7 mutant and were partly restored by transformation with a plasmid carrying the GLC7 gene. Southern blots indicate the presence of a single copy of GLC7 in S. cerevisiae, and gene disruption experiments showed that the GLC7 gene is essential for cell viability. The GLC7 mRNA was identified as a 1.4-kilobase RNA that increases 4-fold at the end of exponential growth in wild-type cells, suggesting that activation of glycogen synthase is mediated by increased expression of protein phosphatase 1 as cells reach stationary phase.     

Genetic deletion of murine SPRY domain-containing SOCS box protein 2 (SSB-2) results in very mild thrombocytopenia

The SSB family is comprised of four highly homologous proteins containing a C-terminal SOCS box motif and a central SPRY domain. No function has yet been ascribed to any member of this family in mammalian species despite a clear role for other SOCS proteins in negative regulation of cytokine signaling. To investigate its physiological role, the murine Ssb-2 gene was deleted by homologous recombination. SSB-2-deficient mice were shown to have a reduced rate of platelet production, resulting in very mild thrombocytopenia (25% decrease in circulating platelets). Tissue histology and other hematological parameters were normal, as was the majority of serum biochemistry, with the exception that blood urea nitrogen (BUN) levels were decreased in mice lacking SSB-2. Quantitative analysis of SSB mRNA levels indicated that SSB-1, -2, and -3 were ubiquitously expressed; however, SSB-4 was only expressed at very low levels. SSB-2 expression was observed in the kidney and in megakaryocytes, a finding consistent with the phenotype of mice lacking this gene. Deletion of SSB-2 thus perturbs the steady-state level of two tightly controlled homeostatic parameters and identifies a critical role for SSB-2 in regulating platelet production and BUN levels.     

Deletion of the Hsp70 chaperone gene SSB causes hypersensitivity to guanidine toxicity and curing of the [PSI+] prion by increasing guanidine uptake in yeast

Yeast Ssb proteins (Ssbp) are ribosome-associated Hsp70 chaperones that function in translation. Elevated levels of Ssbp enhance the ability of over-expressed Hsp104 chaperone to eliminate the yeast [PSI+] prion, while depletion of Ssbp reduces this effect. Millimolar concentrations of guanidine in the growth medium cure yeast cells of prions by inactivating Hsp104. Guanidine is also toxic to yeast, irrespective of the status of Hsp104 and [PSI+]. Strains that lack Ssbp are hypersensitive to guanidine toxicity. Here we show that ssb- cells have normal numbers of [PSI+] "seeds", but can be cured of [PSI+] using one-sixth of the guanidine concentration required to eliminate [PSI+] from SSB cells. Correspondingly, the level of intracellular guanidine was eight-fold higher in ssb- cells than in wild-type cells, which explains all effects of Ssbp depletion on susceptibility to guanidine. The sensitivity of wild-type cells to the effects of guanidine also correlated with guanidine uptake, which was enhanced at low temperature. Guanidine sensitivity of strains mutated in any of 16 ABC membrane transporters, which are implicated in multidrug resistance, was normal. We found that an erg6 mutant that has an altered membrane lipid composition was hypersensitive to guanidine toxicity, but the lipid composition of ssb- cells was identical to that of wild-type cells. Our results suggest that Ssbp depletion does not affect prion seed regeneration, and that elevated guanidine uptake by ssb- cells may be due to increased retention rather than to an alteration in active or passive transport of the compound.     

A mutation in the consensus ATP-binding sequence of the RecD subunit reduces the processivity of the RecBCD enzyme from Escherichia coli.

We have constructed a mutant form of the RecBCD enzyme from Escherichia coli with a lysine to glutamine change in the consensus ATP-binding sequence in the RecD subunit (Korangy, F., and Julin, D.A. (1992a, 1992b) J. Biol. Chem., 1727-1732; 1733-1740). We compare here the kinetics of double-stranded DNA-dependent ATP hydrolysis by the mutant (RecBCD-K177Q) and wild-type enzymes. We included heparin to trap enzyme not bound to DNA, or the single-stranded DNA-binding (SSB) protein from Escherichia coli to prevent the enzyme from binding to single-stranded DNA products and partially single-stranded reaction intermediates. The ATP hydrolysis kinetics in either case show a rapid burst phase followed by a slower second phase. The wild-type enzyme hydrolyzes an amount of ATP about equal to the DNA nucleotide concentration in the rapid phase. The amount of ATP hydrolyzed by the RecBCD-K177Q enzyme in the burst is about 8-10-fold lower than the wild-type, in the presence of either heparin or SSB. The burst magnitude of the wild-type enzyme with heparin is proportional to the size of the DNA from about 1,420 to 22,400 base pairs whereas that of the mutant is independent of the DNA size. The wild-type enzyme completely degrades a 6,250-base pair DNA substrate with no partially degraded molecules visible on agarose gels. RecBCD-K177Q enzyme reaction mixtures in the presence of SSB protein contain a heterogeneous mixture of partially degraded molecules of 2,000-5,000 base pairs. These results indicate that the RecBCD-K177Q enzyme is less processive than the wild-type enzyme.     

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.     

Variable expression of the ssb-1 allele in different strains of Escherichia coli K12 and B: Differential suppression of its effects on DNA replication, DNA repair and ultraviolet mutagenesis

Summary We have transduced the mutant allele ssb-1, which encodes a temperature-sensitive single-strand DNA binding protein (SSB), into several Escherichia coli strains, and have examined colony-forming ability, DNA replication, sensitivity to ultraviolet light (UV) and UV-induced mutability at the nonpermissive temperature. We have found: 1) that the degree of ssb-1-mediated temperature-sensitivity of colony-forming ability and of DNA replication is strain-dependent, resulting in plating efficiencies at 42° C (relative to 30° C) ranging from 100% to 0.002%; 2) that complete suppression of the temperature-sensitivity caused by ssb-1 occurs only on nutrient agar, and not in any other medium tested; 3) that strains in which ssb-1-mediated temperature-sensitivity is completely suppressed show moderate UV sensitivity and normal UV mutability at 30° C, but much more extreme UV sensitivity and drastically reduced UV mutability at 42° C; and 4) that defects in excision repair or in other Uvr⁺-dependent processes are not responsible for most of the UV sensitivity promoted by ssb-1. We discuss our results in relation to the known properties of SSB and its possible role in the induction of DNA damage-inducible (SOS) functions.