Template specificity changes of DNA-dependent RNA polymerase in B. subtilis during sporulation

Previous work has indicated that loss of ability of DNA dependent RNA polymerase, from stationary phase cultures of $\text{B. subtilis}$, to transcribe phage øe DNA was a $\text{sine qua non}$ for sporulation. To ascertain if this change in template specificity was sporulation-specific, we repeated these experiments using a defined sporulation medium. The changes observed previously did not occur in the defined medium although sporulation was normal. The ability of the enzyme to transcribe other DNA templates was also examined. Similar studies were carried out using a polymerase from a rifamycin-resistant, sporulation conditional mutant. The significance of these findings with regard to the regulation of sporulation in $\text{B. subtilis}$ is discussed.     

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.     

Ultraviolet light-induced recombination

Stimulation of transduction in $\text{Escherichia coli}$ by ultraviolet irradiation of the transducing phage P1 requires the $\text{uvrA-uvrB}$ nuclease but not the $\text{uvrC}$ product or DNA polymerase I. It is hypothesized that the first step in “normal” recombination can be bypassed by any procedure generating single-stranded ends of DNA (as, for example, by $\text{uvra-uvrB}$ nuclease activity).     

Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication

A spontaneous mutant of $\text{Bacillus}\text{subtilis}$ resistant to killing by two hydroxyphenylazopyrimidines has been isolated. The DNA polymerase III of this mutant is resistant to inhibition by these drugs. The K_i for 6-(p-hydroxyphenylazo)-uracil (HPUra) is 20 μM, about 40 times higher than the K_i of the wild-type enzyme. The mutant and wild-type polymerases behave similarly during purification, are sensitive to N-ethylmaleimide and to 0.1 M KCl, and have the same K_m for dGTP (0.5 μM). The HPUra inhibition of both enzymes is attenuated competitively by dGTP. We conclude that polymerase III is the target for hydroxyphenylazopyrimidines $\text{in}\text{vivo}$, and since the drugs specifically inhibit replicative DNA synthesis, polymerase III is necessary for DNA replication.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Differential effect of neomycin on DNA dependent -DNA and RNA synthesis in vitro

Neomycin inhibits $\text{in vitro}$ DNA dependent DNA and RNA synthesis catalyzed by DNA polymerase I and RNA polymerase from $\text{E. coli}$. The effect of the antibiotic is more pronounced towards DNA synthesis. The inhibition of DNA synthesis is competitive with template DNA, does not reverse with excess deoxynucleoside triphosphate, Mg²⁺ or enzyme $\text{E. coli}$ DNA polymerase I. Neomycin does not reduce the number of potential 3′ -OH end or primer. It seems to shorten the size of the newly formed polynucleotide.     

Lack of mutagenicity and metabolic inactivation of aphidicolin by rat liver microsomes

In view of the possible utilization of aphidicolin, a specific inhibitor of DNA polymerase α, in the treatment of neoplastic diseases, it seemed important to assess the mutagenic effect of the drug and the possible modification induced by metabolic activation in the liver. This paper shows that aphidicolin lacks mutagenicity in the Ames' $\text{Salmonella}$-microsome test in agreement with our previous observation that it does not induce DNA repair synthesis in HeLa cells. During the studies of mutagenicity we have observed that aphidicolin is converted to inactive derivative(s) by rat liver microsomal oxidases. The reaction is dependent on time and temperature and requires NADP⁺ and glucose-6-P. The metabolites are not mutagenic and they do not induce DNA repair synthesis in HeLa cells. Therefore the possible anti-cancer use of aphidicolin is not hampered by its partial metabolic inactivation in liver. Our results suggest however that aphidicolin will possibly be clinically useful at concentrations higher than those expected from our studies with human DNA polymerase $\text{α}\text{in vitro}$ and human neoplastic cell lines $\text{in vivo}$. The metabolic derivative(s) of aphidicolin is inactive both against cellular DNA polymerase α and Herpes simplex viral DNA polymerase.     

Further characterization of a ribonucleotide-polymerizing enzyme from Histoplasma capsulatum. II. Possible role in cellular metabolism

An enzyme, ribonucleotide polymerase, isolated from the yeast phase of a fungus, $\text{Histoplasma capsulatum}$ has been found to stimulate the incorporation of dTMP in the reaction catalysed by DNA polymerase from $\text{H. capsulatum}$ and $\text{E. coli}$. The stimulation is dependent on the amount of ribonucleotide polymerase added. The data indicate that protein-protein interaction is responsible for the increase in DNA synthesis. It is suggested that ribonucleotide polymerase may be involved in supplying short RNA primers for DNA polymerase.     

A template independent, rifampicin sensitive poly (A).poly (U) synthesizing activity present in Bacillus subtilis.

A template independent poly (A)·poly (U) synthesizing activity has been isolated from $\text{Bacillus subtilis}$. This activity is eluted from a DNA-cellulose column along with DNA-dependent RNA polymerase. The column fractions which exhibit this activity contain RNA polymerase holoenzyme plus a polypeptide which is slightly larger than sigma factor; pure RNA polymerase holoenzyme did not synthesize poly (A)·poly (U). The activity was dependent on the presence of ATP, UTP, and Mn⁺⁺ (Mg⁺⁺ could not substitute), and was inhibited by rifampicin, streptolydigin, and Cibacron Blue. The incorporation of nucleotides was not linear with time, but appeared after a lag period. The results suggest that a modified form of DNA-dependent RNA polymerase analogous to $\text{Escherichia coli}$ holoenzyme II is catalyzing the synthesis of poly (A)·poly (U).     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Characterization of the stimulatory effect of T4 gene 45 protein and the gene 4462 protein complex on DNA synthesis by T4 DNA polymerase

On a variety of single-stranded DNA templates, the overall rate of in vitro DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase is increased about fourfold by addition of the T4 gene $\text{44}\text{62}$ and 45 proteins. Several different methods suggest that this stimulation reflects an increase in the average DNA polymerase “sticking distance”, or processivity, from 800 to about 3000 nucleotides per initiation event. Both the $\text{44}\text{62}$ protein complex and the 45 protein must be present to obtain this effect, and either ATP or dATP hydrolysis is required. Rapid-mixing experiments indicate that the polymerase stimulation is maximized within a few seconds after addition of these “polymerase accessory proteins.”     

Transposon (Tn5)-mediated suppressive integration of ColE1 derivatives into the chromosome of Escherichia coli K12 (dnaA)

While integration of ColE1 had not been observed previously by ordinary suppressive integration, a $\text{dnaA}$ (Ts) $\text{E. coli}$ strain with Tn$\text{5}$ at various sites of the chromosome and ColE1 or its mini-derivative, pAO3, but not pSC101, inserted by the same transposon produced integratively suppressed strains depending on the RecA function. In contrast to Hfr strains made with a stringently controlled plasmid, they contained the plasmid not only in an integrated but in an autonomous state at an amount comparable to the strain containing the plasmid only autonomously. Introduction of a RecA-deficient mutation to the strain with an integrated ColE1 derivative through conjugation failed. This is likely to be due to lethality of such a strain without RecA-dependent excision of the integrated high copy number plasmid or to quantitative deficiency of DNA polymerase I in addition to the $\text{recA}$ mutation.     

Repair of deaminated cytosine residues of DNA: biological significance of the absence of uracil from DNA.

Uracil-DNA glycosylase of $\text{Bacillus}\text{subtilis}$ is involved in repair of deaminated cytosine residues of DNA. Survivals of SPO2 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in $\text{urg}$ mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a $\text{urg}$ mutant is three fold higher than is that of a wild type strain.     

Release of chromatin template restriction in rabbit spermatozoa

The addition of the acidic polymers heparin or polyxanthylic acid to rabbit spermatozoa or sperm heads previously exposed to disulfide reducing agents released sperm DNA template restriction and stimulated high levels of incorporation of DNA precursor into DNA, as assayed with exogenous DNA polymerase. Incorporation did not occur in the presence of DNAase, or in the absence of magnesium ion, any of the four deoxyribonucleotides, or $\text{E. coli}$ DNA polymerase. This represents the first report that spermatozoa can synthesize DNA $\text{in vitro}$.     

Novobiocin and nalidixic acid target proteins in yeast

Novobiocin (and its related drug, coumermycin A₁) and nalidixic acid are specific inhibitors of DNA gyrase in bacteria. These drugs inhibit many enzymatic activities in yeast; such as DNA polymerase activity in crude extracts, $\text{in}$$\text{vitro}$ 2-μm plasmid DNA replication, purified DNA polymerase I and II, and topoisomerase I. Therefore, the inhibition by these inhibitors in yeast is not specific for a particular enzyme.     

Detection of a DNA polymerase β stimulating protein in hela cell nuclear extracts

HeLa cell nuclei contain a protein which stimules the $\text{in}\text{vitro}$ activity of HeLa cell DNA polymerase β, but does not affect the activity of DNA polymerase α and γ. The protein, which binds to both single- and double-stranded DNA, does not possess nuclease activity and is heat stable, surviving 100 degrees C for 10 min. The molecular weight of the protein is approximately 85,000 and evidence is presented that it may exert its stimulatory effect by direct interaction with β-polymerase.     

Regulation of the in vitro synthesis of E. coli ribosomal protein L12.

The $\text{in}\text{vitro}$ DNA dependent synthesis of ribosomal protein L12 and the β subunit of RNA polymerase has been investigated using DNA from a plasmid which contains the genetic information for ribosomal protein L12 and the β subunit of RNA polymerase. This DNA, however, lacks the promoter region and the genetic information for the first 26 amino acids of ribosomal protein L10. It was found that L12 and the β subunit of RNA polymerase are efficiently synthesized $\text{in}\text{vitro}$ from this DNA. These results suggest that L12 and the β subunit of RNA polymerase can be synthesized from a promoter situated within the L10 gene.     

Temperature dependent permeability changes of Bacillus subtilis and incorporation of nucleotides into DNA

Cells of $\text{B. subtilis}$ exposed to temperatures between 0 and 5 C are permeable to small molecules not normally able to pass through the cell envelope. As a result, deoxyribonucleotide triphosphates (dXTPs) are incorporated into DNA if the reaction mixture contains all four dXTPs. Since this incorporation is insensitive to 6-(p-hydroxyphenylazo)-uracil and is not observed in DNA Polymerase I mutants, we conclude it reflects DNA repair rather than the DNA replication which can be observed in cells permeabilized with toluene.