Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids.

Competent cell-deoxyribonucleic acid (DNA) interactions were examined using tritium-labeled homologous or heterologous native or denatured DNAs and competent Streptococcus sanguis Wicky cells (strain WE4). The DNAs used were extracted from WE4 cells, Escherichia coli B cells, and E. coli bacteriophages T2, T4, T6, and T7. The reactions examined were: (i) total DNA binding, (ii) deoxyribonuclease-resistant DNA binding, and (iii) the production of acid-soluble products from the DNA. Optimal temperatures for the reactions were as follows: reaction (i), between 30 and 40 degrees C; reaction (ii), 30 degrees C; and reaction (iii), greater than 40 degrees C. The rates for the reactions (expressed as molecules of DNA that reacted per minute per colony-forming unit) did not vary greatly from one DNA source to another. With a constant competent cell concentration and differing DNA concentrations below a saturation level (from a given source), a different but constant fraction of the added DNA was cell bound, deoxyribonuclease resistant, and degraded to acid-soluble products. In experiments where the number of competent cells was varied and the DNA concentration was held constant, again essentially the same result was obtained. The extent of reactions (i), (ii), and (iii) depended upon the numbers as well as the source of DNA molecules applied to competent cells. Calcium ion essential for native DNA-cell reactions was also found essential for denatured DNA-cell reactions. Data obtained from competition experiments lead to the conclusion that competent WE4 cells contain specific sites for native as well as denatured DNAs.     

Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.     

Cell surface-located deoxyribonucleic acid receptors in transformable pneumococci.

We studied deoxyribonucleic acid (DNA) binding in transformable pneumococci. The relevant findings are as follows. (i) At least half of the DNA Molecules adsorbed to competent cells in the growth medium are attached to sites on the protoplast membrane. (ii) Most of the DNA bound to live competent cells in the presence of glucose is not released by moderate shear or by autolysin treatment. In contrast, most of the DNA adsorbed to competent cells in the absence of glucose is shear and autolysin sensitive. (iii) The presence of binding sites resembling in properties the sites in live competent cells can be demonstrated in wall-membrane complexes. Most of these sites are lost during preparation of cell walls and protoplasts. It is suggested that the DNA-binding site is a membrane component (protein?) Stabilized by polysaccharide (cell Wall) material. (IV) Mechanical or enzymatic damage to the cell wall or change in the ionic conditions can induce DNA binding (and surface-nuclease activity) in the incompetent pneumococci. However, such cells still show neither genetic transformation nor extensive nuclease-resistant binding of DNA. It is suggested that both competent and incompetent cells contain a large number of sequestered DNA-binding sites that can be unmasked by several experimental conditions. Induction of the competent state by the competence activator protein may involve an endogenous unmasking process.     

Quantitive Autoradiographic Study of Competence and Deoxyribonucleic Acid Incorporation in Bacillus subtilis

The relationships among deoxyribonucleic acid (DNA) uptake, transformation, and autoradiographic labeling were investigated. It is shown that: (i) autoradiography is a good method for measuring the total fraction of competent cells able to incorporate transforming DNA; in our best experiment that fraction was 11.5%. (ii) Computation of the fraction of competent cells in a culture of Bacillus subtilis, by comparing the frequencies of single and double transformants for two unlinked markers, gives values which are somewhat different from those obtained by auto-radiography. (iii) On the average, a competent cell of B. subtilis irreversibly takes up from 0.9 x 10(-10) to 1.4 x 10(-10) mug of DNA; this amount represents about 1/55 to 1/35 of the bacterial chromosome weighing 5 x 10(-9) mug, and corresponds to three to six DNA molecules having a molecular weight of 1.6 x 10(7).     

Enchancement of streptococcal transformation yield by proteolytic enzymes.

Trypsin and other proteolytic enzymes, added together with transforming DNA or during cell-DNA contact to competent cultures of several streptococcal strains, enchanced (10 to 600%) the yield of genetic transformation (stimulation). With few exceptions, the level of stimulation was high (over 100%) when competence was low (below 2%). Stimulation was caused by the action of an enzyme on competent cells and not on any other component of transformation mixture. The phenomenon occurred when the enzyme was added to the culture not earlier than 7 min before and not later than 5 min after the period of cell-DNA contact. The presence of trypsin during cell-DNA contact caused: (i) the alterations at cell surface, demonstrated by electron microscopy, increased release of 3H-amino acid-labeled material, and higher cell susceptibility to autolysis; (ii) the increase of both total and irreversible binding of DNA by the cells; and (iii) the decrease of early nucleolytic degradation of DNA by cells. These and other data point to the importance of a delicate balance of recipient cell's surface nuclease activities in the effectiveness of transformation process. It is also possible that trypsin eliminates an unknown cellular factor which obstructs DNA-cell receptors interaction.     

Inhibition of the Development of Competence in Streptococcus sanguis (Wicky) by Reagents that Interact with Sulfhydryl Groups: Discernment of the Competence Process

Reagents that interact with sulfhydryl groups are shown to inhibit competence factor (CF)-induced competence development in Streptococcus sanguis (Wicky) strain WE4 (Wicky 4 Ery(R)). Inhibition is correlated with specific inhibition of either the function or biosynthesis of three competent cell-related proteins and is reversed by either 2-mercaptoethanol or dithiothreitol. Mercuric chloride (5 muM) or N-ethylmaleimide (NEM; 50 muM) inhibited (i) the function but not the biosynthesis or activation of the competent cell-associated autolysin; (ii) the biosynthesis of a competent cell-associated protein of unknown function, demonstrated by polyacrylamide gel electrophoresis of acidified phenol extracts; and (iii) the biosynthesis or activation of distinct deoxyribonucleic acid (DNA)-binding sites. Neither reagent at the indicated concentration interfered with the uptake of CF by cells or with the uptake and expression of DNA by competent cells. Neither reagent inactivated CF or genetic markers coded by the transforming DNA, nor did they inhibit cell growth or viability appreciably. The data reveal that either mercuric chloride or NEM can differentially inhibit induced protein synthesis and, in addition, conclusively show that some autolytic activity is essential for the onset of the competent state.     

Number of Deoxyribonucleic Acid Uptake Sites in Competent Cells of Bacillus subtilis

Two direct methods are presented for estimating the average number of deoxyribonucleic acid (DNA) uptake sites in competent cells of Bacillus subtilis from measurement of (14)C- or (3)H-thymine-labeled DNA uptake by competent culture. Advantage is taken of two facts: (i) effective contact between competent cells and transforming DNA molecules is established within a short time after mixing them together, and (ii) DNA molecules enter the competent B. subtilis cells in a linear fashion at a finite speed. From the number of DNA molecules initially attached to competent cells by brief exposure to transforming DNA in the first method or from the rate of DNA uptake by competent culture in the second method, the average number of DNA uptake sites is calculated to be 20 to 53 per competent cell.     

Purification and Properties of Deoxyribonucleic Acid Binding Factor Isolated from the Surface of Streptococcus sanguis Cells

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S₁ action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.     

Different nuclease activities in competent and noncompetent Bacillus subtilis.

Competent and noncompetent cells of Bacillus subtilis were separated on the basis of their different buoyant densities. The two types of cells were compared with respect to their interactions with exogenous deoxyribonucleic acid(DNA). After exposure of DNA to the cells, the unadsorbed fraction of DNA molecules was examined. Both types of cells decreased the biological activity of this DNA, the inactiviation exerted by noncompetent cells being more severe than that exerted by competent cells. Sedimentation analysis of the inactivated DNA revealed that fragments of DNA are produced, owing mainly to the introduction of double-strand scissions. In addition to this fragmentation, the competent bacteria extensively digested the DNA exonucleolytically. This type of breakdown was specifically related to the competent state rather than to the state of low density. The exonucleolytic activity is, in all probability, associated with the cell envelope, because most of the activity is released into the medium when the cells are converted to protoplasts. At 37 C the competence-specific exonucleolytic breakdown started 2 to 3 min after the binding of DNA to the cells. In unfractionated cultures, breakdown may proceed until 70% of the total amount of DNA added has been made acid soluble. Nontransforming Escherichia coli DNA was also subject to exonucleolytic degradation; it seems unlikely,therefore, that this type of breakdown occurs as a consequence of recombination. Since ethylenediaminetetraacetate blocked both transformation by native DNA and the exonucleolytic breakdown of bound DNA, we suggest that the breakdown of DNA by competent cells fulfills an essential function in genetic transformation of B. subtilis.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.     

ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA.

Binding of adenosine (3-thiotriphosphate) (ATPgammaS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) was studied by filter binding. RFC alone bound 1.8 ATPgammaS molecules. However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPgammaS molecules, respectively, were bound. When both PCNA and DNA were present 3.6 ATPgammaS molecules were bound per RFC. Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA.PCNA.RFC complex. An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC.DNA complex dissociated with hydrolysis of ATP. Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC.ATP(2)); (ii) this complex binds PCNA (PCNA.RFC.ATP(2)), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA.RFC.ATP(3)); (iii) this complex can bind DNA to yield DNA.PCNA.RFC.ATP(3); (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA.PCNA.RFC.ATP(4) complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.     

Quantitative analysis of internalization of glucagon by isolated hepatocytes

Biochemical methods have been used to quantitate total, acid-stable and acid-labile association of (mono[125I]iodoTyr10) glucagon with rat hepatocytes in suspension to evaluate internalization of glucagon and its receptors. Internalization is inhibited by low temperature, phenylarsine oxide, and by blocking receptor binding, consistent with receptor-mediated endocytosis. Approximately 30% of the total cell-associated hormone is internalized at 30 min of incubation. The rate declines until 90 min when the internalization of glucagon ceases, although the cells remain competent to internalize asialofetuin. From 90 min to 4 h, 27% of the maximum label internalized at 30 min remains within cells. The number of cell surface receptors decreases but the affinity of those remaining is unchanged. However, 1.7-2.7 surface receptors are lost to binding for each molecule of radiolabeled glucagon internalized. Uptake occurs according to a rate constant of 0.183 min-1 (t1/2 = 3.8 min). We conclude that (i) hepatocytes internalize a finite quantity of glucagon, implying the existence of undefined regulatory mechanisms; (ii) hormone is retained for greater than 2 h within cells and may play a physiological role within cells; and (iii) both occupied and unoccupied receptors become inaccessible to extracellular hormone as internalization proceeds; rapid recycling of receptors does not occur.     

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.     

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.     

Mode of Action of the Competence-Inducing Factor of Bacillus stearothermophilus

Development of competence in the noncompetent mutant of Bacillus stearothermophilus 4S requires a soluble competence-inducing factor (CF). This study describes several observations that define the mechanism of action of this factor. (i) CF interacts with cells, protoplasts, or cytoplasmic membranes in a temperature-dependent reaction; incubation of CF with cells alters the sedimentation properties of the cells. (ii) Association between CF and the cytoplasmic membrane appears to be the first and rate-limiting step in the process of transfection with deoxyribonucleic acid (DNA) from bacteriophage TP-IC. (iii) The membrane-CF complex is capable of removing infectious DNA from the supernatant fraction in vitro. (iv) Twenty phage-resistant mutants are unable to undergo the above reactions. A model is developed in which CF serves as a dynamic mediator of the reaction between the transfecting DNA and the cell surface.     

Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.