SEARCH · PubMed Health

Results for “SSB”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources

PubMed1995

Differences in the mechanism of stimulation of T7 DNA polymerase by two binding modes of Escherichia coli single-stranded DNA-binding protein.

Escherichia coli single-stranded DNA-binding protein (Eco SSB) has been shown previously to display several DNA binding modes depending on the ionic conditions. To determine what effect these various binding modes have on DNA replication, we have studied DNA synthesis by the T7 DNA polymerase under ionic conditions where Eco SSB interacts with either 72 or 91 nucleotides of M13 DNA. These forms presumably correspond to the previously described (SSB)56 and (SSB)65 (Lohman and Ferrari, 1994) that were determined using the binding of SSB to homopolymers. Here we report the stimulation induced by (SSB)91 to be 4-fold greater than that produced by (SSB)72 under conditions where the template is in large excess. Surprisingly, when the polymerase level is raised so that it is in molecular excess, (SSB)91 no longer stimulates synthesis while (SSB)72 affords a 4-fold stimulation, which is the same level of stimulation as when the template was in excess. Both SSB forms increase the rate of DNA synthesis and were found to stimulate synthesis by relieving template secondary structures. However, (SSB)72 specifically increases strand displacement synthesis, while (SSB)91 stimulates synthesis by increasing the affinity of the polymerase for the template.

Base Sequence↗

PubMed1994

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

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

Allosteric Regulation↗

PubMed1989

Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. I. Evidence and a quantitative model.

The interaction of the Escherichia coli single strand binding (SSB) protein with single-stranded DNA is complex, since a number of different binding modes have been observed, with different DNA site sizes and binding properties and the transitions among these binding modes are strongly influenced by solution conditions in vitro. Recent experiments have suggested the existence of negative co-operativity among the multiple DNA binding sites within individual SSB tetramers. In order to probe this negative co-operativity, we have examined the binding of a series of oligonucleotides of varying length, using the quenching of the intrinsic SSB protein fluorescence to monitor binding. The stoichiometries for saturation of the SSB tetramer are 4, 2, 2, 1 and 1, for the oligonucleotides, dT(pT)N-1, with N = 16, 28, 35, 56 and 70, respectively, indicating that one molecule of either dT(pT)27 or dT(pT)34 interacts with two SSB subunits, whereas one molecule of dT(pT)15 interacts with only a single subunit. Saturation of the SSB tetramer with dT(pT)15, dT(pT)34, dT(pT)69 or poly(dT) results in 85 to 90% quenching of the SSB fluorescence, whereas saturation with dT(pT)27 or dT(pT)55 results in only 80% and 72% quenching, respectively. Therefore, a single-stranded DNA of at least 64 nucleotides is required to wrap around an SSB tetramer fully and interact with all four subunits. A quenching of 50(+/- 2)% is observed upon filling only half of the subunits with either one molecule of dT(pT)34 or two molecules of dT(pT)15, which agrees with the quenching and site size observed in the (SSB)35 polynucleotide binding mode. Direct binding measurements indicate that the binding of dT(pT)27 to its second site is influenced by the oligonucleotide that occupies the first binding site (either dT(pT)27 or dT(pT)34), providing proof for the existence of a true negative co-operativity. This negative co-operativity is observed also for the binding of the shorter oligonucleotide, dT(pT)15. A statistical thermodynamic ("square") model gives an excellent description of the binding of all oligonucleotides possessing multiple sites on the SSB tetramer, based on only two interaction constants, the intrinsic binding constant, KN, and the negative co-operativity parameter, sigma N. These data indicate that the binding sites (subunits) on the unliganded SSB tetramer are all equivalent, but that a non-equivalence between dimers of subunits within the tetramer is induced upon binding ssDNA.

Bacterial Proteins↗

PubMed2000

Distinct properties of Mycobacterium tuberculosis single-stranded DNA binding protein and its functional characterization in Escherichia coli.

Single-stranded DNA binding proteins (SSBs) play an essential role in various DNA functions. Characterization of SSB from Mycobacterium tuberculosis, which infects nearly one-third of the world's population and kills about 2-3 million people every year, showed that its oligomeric state and various in vitro DNA binding properties were similar to those of the SSB from Escherichia coli. In this study, use of the yeast two-hybrid assay suggests that the ECO:SSB and the MTU:SSB are even capable of heterooligomerization. However, the MTU:SSB failed to complement a Deltassb strain of E. coli. The sequence comparison suggested that MTU:SSB contained a distinct C-terminal domain. The C-terminal domain of ECO:SSB interacts with various cellular proteins. The chimeric constructs between the N- and C-terminal domains of the MTU:SSB and ECO:SSB exist as homotetramers and demonstrate DNA binding properties similar to the wild-type counterparts. Despite similar biochemical properties, the chimeric SSBs also failed to complement the Deltassb strain of E.coli. These data allude to the occurrence of a 'cross talk' between the N- and the C-terminal domains of the SSBs for their in vivo function. Further, compared with those of the ECO:SSB, the secondary/tertiary interactions within MTU:SSB were found to be less susceptible to disruption by guanidinium hydrochloride. Such structural differences could be exploited for utilizing such essential proteins as crucial molecular targets for controlling the growth of the pathogen.

Amino Acid Sequence↗

PubMed1996

The use of the single cell gel electrophoresis assay in detecting DNA single strand breaks in lung cells in vitro.

DNA single strand breaks (SSB) can be used as a biomarker of oxidant exposure, and also as an indicator of the carcinogenicity/ mutagenicity of a substance. The single cell gel electrophoresis (SCGE) assay is more sensitive and requires fewer cells compared to other techniques used for detecting SSB. We examined the utility of using the SCGE assay for human lung cells exposed to endogenous and exogenous oxidants. A human bronchial cell line (BEAS) was used as a model of airway epithelial cells in this study. BEAS cells exposed to 0-50 microM hydrogen peroxide (H2O2) for 60 min at 4 degrees C exhibited a concentration-dependent increase in SSB as determined by an increased DNA migration area in a gel undergoing electrophoresis. H2O2-induced increases in DNA SSB were also demonstrated using cultured normal human tracheobronchial epithelial (NHBE) cells and human alveolar macrophages in a concentration response manner. BEAS cells were also exposed to air or ozone (O3) on a Transwell filter without medium present apically. Cells exposed to O3 at 0.1 or 0.4 ppm at 37 degrees C for up 120 min had a time- and concentration-dependent increase in SSB compared to air-exposed cells. NHBE cells exposed to 0.4 ppm O3 (60 min) also had increased DNA SSB. Cells with H2O2-induced DNA SSB can be frozen and stored up to 4 weeks without altering the original DNA SSB. These findings indicate that SCGE can be used to detect SSB in cultured lung cells, and has applicability for detecting SSB in lung cells recovered from in vivo and in vitro exposures to oxidants.

Bronchi↗

PubMed1981

Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4'-(9-acridinylamino) methanesulfon-m-anisidide and adriamycin.

The DNA intercalating agents 4'-(9-acridinyl-amino) methanesulfon-m-anisidide (m-AMSA) and adriamycin were studied by using filter elution methods to measure DNA single-strand breaks (SSB's), DNA-protein cross-links (DPC's), and double-stranded breaks (DSB's) in mouse leukemia L1210 cells. Both compounds produced SSB's and DPC's at nearly 1:1 ratios. The SSB's and DPC's were shown to be localized with respect to each other; this was inferred from the finding that filter assays based on protein adsorption completely prevented the elution of the DNA single-strand segments between SSB's. In the case of m-AMSA, which produces relatively high frequencies of DNA lesions, the possibility that a protein bridges across the SSB was excluded by alkaline sedimentation studies. Both compounds also produced DSB's, but the SSB/DSB ratios differed; the SSB/DSB ratios increase in the following order: ellipticine greater than adriamycin greater than m-AMSA greater than X-ray [results of this paper combined with those of Ross, W. E., & Bradley, M. O. (1981) Biochim. Biophys. Acta (in press)]. The o-AMSA isomer is much less cytotoxic than m-AMSA and did not produce protein-associated strand breaks. The simplest model to explain the results is that a protein becomes covalently bound to either the 3' or the 5' termini of the intercalator-induced strand breaks. At moderately cytotoxic doses, m-AMSA yielded much larger frequencies of protein-associated SSB's than did adriamycin. m-AMSA-induced protein-associated SSB's saturated at approximately 60000 per cell over a concentration range in which m-AMSA uptake by the cells was proportional to the drug concentration. m-AMSA was found to enter and exit from cells very rapidly at 37 degrees C; protein-associated SSB's and DSB's also appeared and disappeared rapidly. At reduced temperature, however, the appearance and disappearance of protein-associated SSB's could be blocked while m-AMSA entry and exit still occurred. The saturation behavior and temperature dependence suggest that the formation and disappearance of protein-associated strand breaks is enzymatic. The simplest hypothesis is that the linked protein is a nuclease, such as a topoisomerase, which becomes bound to one terminus of the strand break it produces. It is proposed that topoisomerases producing SSB's and DSB's are stimulated to different degrees by different intercalators.

Aminoacridines↗

PubMed1997

Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants.

A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(T)32, were observed for native SSB, SSB1-135, SSB1-115, and SSB37-177, suggesting that the (SSB)65 and (SSB)35 binding modes, as previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.

Binding Sites↗

PubMed1996

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination.

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor.

Bacterial Proteins↗

PubMed1997

Structural features of phi29 single-stranded DNA-binding protein. II. Global conformation of phi29 single-stranded DNA-binding protein and the effects of complex formation on the protein and the single-stranded DNA.

The strand-displacement mechanism of Bacillus subtilis phage phi29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, phi29 SSB, to be replicated in vivo. To understand the characteristics of phi29 SSB-ssDNA complex that could explain the requirement of phi29 SSB, we have (i) determined the hydrodynamic behavior of phi29 SSB in solution and (ii) monitored the effect of complex formation on phi29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 +/- 0.1) x 10(8) gs(-1), and its rotational correlation time, 7.0 +/- 0.5 ns, phi29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that phi29 SSB is highly organized within a wide range of temperatures (15 to 50 degrees C), being mainly constituted by beta-sheet elements (approximately 50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of phi29 SSB.

Bacillus Phages↗

PubMed1992

Iron mobilization from crocidolite asbestos greatly enhances crocidolite-dependent formation of DNA single-strand breaks in phi X174 RFI DNA.

The ability of the iron associated with asbestos to catalyze damage to phi X174 RFI DNA was determined and compared with iron mobilized from asbestos. Asbestos (1 mg/ml) suspended for 30 min in 50 mM NaCl containing 0.5 micrograms phi X174 RFI DNA, pH 7.5, did not catalyze detectable amounts of DNA single-strand breaks (SSB). However, addition of ascorbate (1 mM) resulted in 19, 26, 7 or 8% DNA with SSB for crocidolite, amosite, chrysotile or tremolite respectively. The percentage of DNA with SSB induced by each form of asbestos was directly related to its iron content. Inclusion of desferrioxamine B, which binds Fe(III) rendering it redox inactive, completely inhibited asbestos-dependent formation of DNA SSB, suggesting that iron was responsible for catalyzing the formation of DNA SSB. Mobilization of Fe(II) from crocidolite by citrate, EDTA or nitrilotriacetate (1 mM) in the absence of ascorbate resulted in 15, 33 or 63% DNA with SSB respectively. This activity was completely inhibited by compounds considered to be .OH scavengers, i.e. mannitol, 5,5-dimethyl-1-pyrroline N-oxide or salicylate (100 mM). Preincubation of crocidolite with citrate (1 mM) for 24 h resulted in mobilization of 52 microM iron and increased ascorbate-dependent induction of DNA SSB compared with crocidolite that was preincubated without citrate. Iron mobilized by citrate was entirely responsible for crocidolite-dependent formation of DNA SSB as evidenced by complete inhibition with desferrioxamine B. Therefore, the results of the present study strongly suggest that iron was responsible for asbestos-dependent generation of oxygen radicals, which resulted in the formation of DNA SSB. Mobilization of iron by chelators, followed by redox cycling, greatly enhanced crocidolite-dependent formation of DNA SSB. Thus, mobilization of iron in vivo by low mol. wt chelators may lead to the increased production of reactive oxygen species resulting in damage to biomolecules, such as DNA.

Asbestos↗

PubMed1991

Formation of single and double strand breaks in DNA ultraviolet irradiated at high intensity.

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.

Coliphages↗

PubMed1987

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.

DNA, Single-Stranded↗

PubMed2006

Murine homologues of the Drosophila gustavus gene are expressed in ovarian granulosa cells.

Mammalian homologues of genes that control oogenesis in other organisms may play similar roles in mammalian ovarian development. In Drosophila melanogaster, GUSTAVUS (GUS) protein physically interacts with and is necessary for the proper posterior localization of VASA protein, and thus is required for specification of germ cells. We identified two mouse genes, SSB-1 and SSB-4 (SPRY domain SOCS box protein), whose protein products share 75% identity and are each approximately 70% identical to Drosophila GUS. Both SSB-1 and SSB-4 mRNA were detectable in mouse ovaries by Northern blotting of total and poly(A) + RNA, but were expressed in few other tissues. SSB-1 was detectable in testes, although the 3'-untranslated region of the mRNA was considerably shorter than the ovarian mRNA. In situ hybridization and RT-PCR analysis of ovaries revealed that both genes were expressed in granulosa cells at all stages of follicular development. In contrast, expression was barely detectable in in oocytes. Immunoblotting analysis revealed that SSB-1 protein was present in follicles at different stages of growth, and immunocytochemistry confirmed that SSB-1 and SSB-4 were detectable in granulosa cells of primary and subsequent stage follicles and that they were present in both mural and cumulus granulosa cells of antral follicles. These results establish that GUS-related proteins, which in Drosophila are restricted to the germ cells, are in the mouse instead expressed in the granulosa cells and are present throughout folliculogenesis. Based on their tissue-restricted pattern of expression and apparent abundance in granulosa cells, we propose that SSB-1 and SSB-4 play key roles in regulating granulosa cell physiology.

Amino Acid Sequence↗

PubMed1985

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

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

DNA, Bacterial↗

PubMed1990

A comparison of the techniques of alkaline filter elution and alkaline sucrose sedimentation used to assess DNA damage induced by 2-nitroimidazoles.

The induction of DNA single-strand breaks (DNA-SSB) in Chinese hamster V79-379A lung fibroblasts by misonidazole or RSU-1069 under both aerobic and hypoxic conditions was examined following incubations for up to 4 hr at 310 degrees K using the technique of alkaline filter elution. Incubation with RSU-1069 induces DNA-SSB under both hypoxic and aerobic conditions, whereas incubation with misonidazole induces DNA-SSB only under hypoxia. The yield of breaks is dependent on both agent concentration and contact time. Following identical treatments with these agents, the yield of DNA-SSB (expressed in radiation dose equivalents) determined by alkaline filter elution is about one order of magnitude less than that previously determined by alkaline sucrose gradient sedimentation. In contrast to radiation induced DNA-SSB, alkaline elution is less sensitive than alkaline sucrose gradient sedimentation when determining DNA-SSB induced by RSU-1069 and misonidazole. During the filter elution assay, either increasing cell lysis from 2 to 4 hr, the pH of the lysing buffer from pH 8.7 to 12.5 or the elution buffer from pH 12.2 to 12.5 does not significantly effect the yield of DNA-SSB. Increasing the pH of the lysing or elution buffers to greater than pH 13 however results in considerable degradation of the DNA, whereby 50-85% of the total DNA passes through the filter with the lysing solution. This effect was similar for DNA from both control and chemically insulted cells. In conclusion, it is apparent that incubation with these agents results in the induction of DNA damage which is expressed as a DNA-SSB only after prolonged treatment under alkaline conditions. Further, the use of alkaline elution to study DNA-SSB damage induced chemically must be treated with caution in the light of these findings.

Animals↗

PubMed1982

Influence of single-stranded DNA-binding protein on recA induction in Escherichia coli.

Two ssb mutants of Escherichia coli, which carry a lesion in the single-strand DNA-binding protein (SSB), are sensitive to UV-irradiation. We have investigated the influence of SSB on the "SOS" repair pathway by examining the levels of recA protein synthesis. These strains fail to induce normal levels of recA protein after treatment with nalidixic acid or ultraviolet light. The level of recA protein synthesis in wild-type cells is about three times greater than ssb cells. This deficiency in ssb mutants occurs in all strains and at all temperatures tested (30-41.5 degrees). In contrast, the ssb-1 mutation has no effect on temperature-induced recA induction in a recA441 (tif-1) strain. Cells carrying ssb+ plasmids and overproducing normal DNA-binding protein surprisingly are moderately UV-sensitive and have reduced levels of recA protein synthesis. Together these results establish that single-strand DNA-binding protein is involved in the induction of recA, and accounts, at least in part, for the UV sensitivity of ssb mutants. Three possible mechanisms to explain the role of SSB are discussed.

Bacterial Proteins↗

PubMedDate not supplied

Lung and cardiac tissue doses in left breast cancer patients treated with single-source breast brachytherapy compared to external beam tangent fields.

PURPOSE: We performed a retrospective analysis of doses to normal critical structures in 26 women with left-sided breast tumors treated with a high-dose-rate single source brachytherapy (SSB) system, versus whole breast external beam tangent fields (EBTF) using a computer model. METHODS AND MATERIALS: Each patient's EBTF radiation plan consisted of ipsilateral tangent fields normalized to 46 Gy. A complementary SSB radiation plan was designed to deliver 34 Gy in 10 fractions at 1cm into tissue enclosing a sphere of diameter 4.6 cm at tumor bed, simulating our institution's experience with the MammoSite applicator. Critical structure volumes and doses analyzed included the volume of whole heart and left ventricle receiving >25 Gy, and left lung (LL) receiving 20 and 13 Gy for EBTF plans. For SSB we determined the linear quadratic 2-Gy-equivalent high-dose-rate doses of 23.4, 20, and 14 Gy to be equivalent to 25, 20, and 13 Gy, respectively. We then compared the two doses for each critical structure. RESULTS: The percent of whole heart receiving >25 Gy equivalent using EBTF and SSB was 2% and 0% (mean dose 2.52 vs. 1.65 Gy), left ventricle receiving >25 Gy using EBTF and SSB was 3% and 0% (mean dose 3.78 vs. 2.68 Gy), LL receiving >20 Gy using EBTF and SSB was 11.5% and 0.5%, and LL receiving >13 Gy using EBTF and SSB was 12.6% and 1.85% (mean dose to LL was 6.06 Gy for EBTF vs. 2.25 Gy for SSB), respectively. CONCLUSIONS: When compared to EBTF, breast brachytherapy with SSB is associated with a significant decrease in mean dose and percentage volume receiving a given dose for each organ.

Brachytherapy↗

PubMed1989

Purification and characterization of a protein from Saccharomyces cerevisiae that binds tightly to single-stranded DNA and stimulates a cognate strand exchange protein.

A single-stranded DNA binding protein (yeast SSB protein) was purified to near-homogeneity from mitotic Saccharomyces cerevisiae cells. The Mr 34,000 protein specifically eluted at high salt (approximately 1200 mM NaCl) during chromatography on a single-stranded DNA-cellulose column. The protein formed stable complexes with single-stranded DNA in an apparent cooperative fashion. As judged from titration and competition experiments, the affinity of the protein was much higher for single-stranded DNA than for double-stranded DNA or single-stranded RNA. The SSB protein also was found to stimulate the strand exchange reaction between linear M13mp19 RF DNA and circular M13mp19 viral DNA as catalyzed by a yeast strand exchange protein previously purified in this laboratory [Kolodner, R., Evans, D. H., & Morrison, P. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5660-5664]. Titration experiments showed maximum stimulation of joint molecule formation at a stoichiometry of about 1 Mr 34,000 monomer yeast SSB per 18 nucleotides of single-stranded DNA. Kinetic experiments demonstrated at least an 18-fold increase in the rate of strand exchange due to the presence of the SSB in reactions where the amount of strand exchange protein was limiting. The yeast SSB protein stimulated the Escherichia coli RecA protein in the strand exchange reaction involving linear M13mp19 RF DNA and circular M13mp19 viral DNA as efficiently as E. coli SSB. However, the E. coli SSB protein did not substitute for the yeast SSB protein in reactions with the yeast strand exchange protein. This suggests that the stimulation of the yeast strand exchange protein by the yeast SSB may involve specific protein/protein interactions.

Chromatography, Affinity↗