The effects of thioketo substitution upon uracil-adenine interactions in polyribonucleotides. Synthesis and properties of the alternating polynucleotides poly(r(A-s2U)) and poly(r(A-s2s4U)).

The polynucleotides poly[r(A-s-2U)] and poly]r(A-s2s4U)] have been synthesized and characterized by nearest-neighbour analysis, sedimentation analysis as well as spectroscopic techniques. Absorption-temperature profile and absorption-pH profile of poly[r(A-s-2U)] did not reveal a structural transition between 10 and 95 degrees C even at low ionic strength, although a variety of properties indicated a helical structure of poly[r(A-s-2U)]: remarkable hyperchromicity of the absorption spectrum, circular dichroic spectrum displaying extrema of large amplitudes, resistance against hydrolysis by ribonuclease and interaction with ethidium bromide in a manner which is characteristic of helical polynucleotides. Our results show that interactions of the type A-s-2U and A-s-2s-4U do in fact exist in helical polynucleotides. The properties of poly]r(As-2U)] furthermore demonstrate the general stabilizing effect of 2-thioketopyrimidine bases in helical polynucleotides by virtue of vertical stacking interactions with neighbouring pyrimiding and purine bases.     

Conformational changes of nucleic acids and poly (d(A-T)-d(A-T)) caused by photoaddition of furocoumarins.

DNA's of various AT content, poly[d(A-T)-d(A-T)], and double-stranded RNA were irradiated with UV light at 365 nm in the presence of linear (xanthotoxin) or angular (angelicin) furocoumarins. The covalent photobinding is strongly dependent on the spatial arrangement of furocoumarin molecules at the polymer conformation. CD measurements demonstrate that the bifunctional photochemical binding of xanthotoxin with double-stranded DNA's and poly[d(A-T)-d(A-T)] is accompanied by conformational changes which involve probably decreasing helical twisting of the double helix. This effect is greatly enhanced with increasing AT content. The formation of A-like structures is very unlikely since the B leads to A transition induced by ethanol addition was found to be strongly suppressed in xanthotoxin photoreacted DNA. The B-type helix appears to be the most sensitive conformation with minor restriction to produce photochemically induced cross-links.     

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase

The steady-state kinetic parameters of the tripeptides D-Val-Leu-Lys-, Ala-Phe-Lys-, and < Glu-Phe-Lys- in which the free carboxyl group was substituted with p-nitroaniline (substrate) or chloromethane (inhibitor), towards the serine proteinases plasmin (EC 3.4.21.7), thrombin (EC 3.4.21.5), urokinase, factor Xa, and trypsin (EC 3.4.21.4) were investigated. The p-nitroanilide derives were found to be very good substrates for plasmin, 2.5--40-times less efficient towards trypsin and very poor (100--10 000-times less efficient) substrates for thrombin, factor Xa and urokinase. The chloromethyl ketone derivatives were comparably efficient inhibitors of plasmin and trypsin and in general very poor (100--10 000-times weaker) inhibitors of thrombin, factor Xa and urokinase. D-Val-Leu-Lys-pNA however was a very poor substrate but D-Val-Leu-Lys-CH2Cl a very efficient inhibitor for thrombin. The variability in susceptibility of the substrates towards the enzymes was due to differences in their Michaelis constant, in their deacylation rate constant or both. the variable efficiency of the inhibitors was mostly due to differences in their dissociation constant and much less to differences in their alkylation rate constant. Only a poor correlation (r = 0.25) was found between the efficiency of the p-nitroanilides as substrate and their homologous chloromethyl ketones as inhibitor. The most notable discrepancy was observed with the D-Val-Leu-Lys derivatives towards thrombin.     

ATP-independent, DNA synthesis by DNA polymerase II in toluenized Bacillus subtilis

Phleomycin stimulates ATP-independent DNA repair synthesis by polymerase II in toluenized $\text{B. subtilis}$ cells. In the presence of ATP it also increases the synthesis, with BrdUTP, of DNA with a density between that of normal DNA and hybrid DNA, and it enhances replicative DNA synthesis by polymerase III.     

Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis

The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected. The amount of poly(A)-RNA during the outgrowth and first cell division remains constant. Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B. subtilis spores.     

Microcalorimetric investigation of dilute and concentrated solutions and films of the polydeoxynucleotide poly(d(A-T)-d(A-T))

Microcalorimetric heat capacity measurements on dilute and concentrated solutions and films of poly[d(A-T)·d(A-T)] in 2 M sodium chloride have been carried out. Values for enthalpy, entropy, and temperature of the helix–coil transition have been found to depend on the polymer concentration, and to have maxima near 20% (w/w) of polymer. The results are discussed in terms of polynucleotide hydration as one of the structure stabilizing factors.     

A novel DNA polymerase induced by Bacillus subtilis phage phi 29.

A novel DNA polymerase induced by Bacillus subtilis bacteriophage phi 29 has been identified. This polymerase can be separated from host DNA polymerase, by fractionation of extracts prepared from phage infected cells, using phosphocellulose chromatography. The isolated polymerase prefers poly(dA)oligo(dT) as template. The DNA polymerase isolated from the cells infected with a gene 2 temperature sensitive mutant (ts2) showed greater heat-lability than that induced by wild type phi 29. The ts2 DNA polymerase was also thermolabile for its activity in the formation of a covalent complex between phi 29 terminal protein and dAMP, the initiation step of phi 29 DNA replication. These findings indicate that gene 2 is the structural gene for a phi 29 DNA polymerase required for the complex formation step of DNA initiation.     

Overproduction and purification of Bacillus subtilis DNA polymerase III.

The objectives of this work were to engineer the cloned polC gene encoding Bacillus subtilis DNA polymerase III for controlled overexpression in Escherichia coli and to devise a facile purification scheme permitting the large-scale production of pure recombinant polymerase. The translational signals of polC were restructured by expression cassette PCR (MacFerrin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1937-1941), and the modified gene was inserted into the expression plasmid, pKC30 (Rosenberg et al., 1983, in "Methods in Enzymology," Vol. 101, pp. 123-138, Academic Press, San Diego), under the strict control of the coliphage lambda pL promoter and its repressor, cI. When the system was derepressed at 32 degrees C, soluble DNA polymerase III accumulated at levels approximating 2% of total cellular protein. The recombinant protein was purified to greater than 99% purity by utilizing a tandem combination of Cibacron blue agarose, phenyl-Sepharose, and MonoQ FPLC chromatography. The properties of the purified recombinant protein were indistinguishable from those of native B. subtilis DNA polymerase III.     

Properties of a Bacillus subtilis strain lacking DNA polymerase I

We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA⁺ toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe₁₂ and argA₃ genes.     

Chelation of calcium ions by poly(gamma-glutamic acid) from Bacillus subtilis(chungkookjang)

Many studies have clarified that poly(gamma-glutamic acid) (PGA) increases the solubility of Ca(2+), suggesting that PGA enhances calcium absorption in small intestine. However, there has been no report on the specific interaction between PGA and Ca(2+) in water. We studied the aqueous solution properties of PGA calcium salt (PGA-Ca complex). The chelating ability and binding strength of PGA for Ca(2+) were evaluated. PGA-Ca complex was soluble in water in contrast with the insolubility of poly(acrylic acid) (PAA) calcium salt and the chelating ability of PGA for Ca(2+) was almost the same than that of PAA. The globular conformation of PGA-Ca complex in water was estimated by SEC and viscosity measurements. The chelation of PGA for Ca(2+) was examined by 1H NMR. The present study showing the characteristics of PGA-Ca complex will provide useful information of the calcium absorption by PGA in vivo.     

Synthesis of poly d(G-C) oligonucleotides

Model sequences for evaluation of the GC dimer sequence repetition on synthesis success were prepared and analyzed by HPLC. Contiguous d(G-C) or d(C-G) sequences have a deleterious effect on DNA oligonucleotide synthesis. The critical number seems to be about 6 GCs in a row. If the GCs are separated by other nucleotides, the effect is not as severe.     

Circular dichroism and thermal melting differentiation of Hoechst 33258 binding to the curved (A(4)T(4)) and straight (T(4)A(4)) DNA sequences

The ability of the B-DNA minor groove ligand Hoechst 33258 to discriminate between prototype curved and straight duplex DNA sequences was investigated by circular dichroism (CD) titrations at the wavelengths of absorbance of the ligand. The sequences were studied either within the framework of the ligated decamers (CA(4)T(4)G)(n) and (CT(4)A(4)G)(n), or within that of the single dodecamers GCA(4)T(4)GC and GCT(4)A(4)GC, to confirm and extend our earlier results based on fluorescence titrations of ligated decamers. A unique, strong binding site is invariantly present in both sequence units. The binding affinity of the drug for the site in the curved A(4)T(4) sequence was found 3- to 4-fold higher compared to the straight sequence. All these features hold true irrespective of the sequence framework, thus confirming that they reflect specific properties of the binding to the two sequences. Ligand binding increases the thermal stability of straight and curved duplex dodecamers to the same extent, thus maintaining the melting temperature differential between the two sequences. However, the different melting patterns and the difference between [total ligand]:[site] ratios needed for site saturation in the two duplexes are in agreement with the difference between binding constants derived from CD measurements.     

Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III.

Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.     

Structure of poly d(A).poly d(T).

On the basis of the x-ray data from polycrystalline and well oriented fibers of the sodium salt of poly d(A).poly d(T) (Arnott et al, Nucl. Acids Res. 11, 4141-4155 (1983), a revised B'-DNA model incorporating B-like adenine and thymine strands is shown to give a much better x-ray agreement (R = 0.25) than the previously assigned model consisting of mixed sugar conformations in the two strands. The narrowing of the minor and the widening of the major grooves are promiscuous features of B'-DNA, which are common to all poly d(purine).poly d(pyrimidine) duplexes with two hydrogen bonded base-pairs and are in marked contrast with classical B-DNA. Due to modest propeller (-15 degrees), the cross strand diagonal hydrogen bonds (0.37 nm) in this duplex are not as strong as those in A,T-rich oligonucleotide crystal structures.