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.     

A novel single strand endonuclease specific for ØX174 DNA

A highly specific endonuclease activity, presumably involving one or both of the products of the ØX174 gene A, has been isolated from ØX174-infected $\text{E. coli}$ by DNA-cellulose chromatography. The enzyme is not present in uninfected cells and binds extremely tightly to DNA-cellulose. It extensively degrades ØX174 viral DNA but does not degrade the circular or linear forms of single stranded viral DNA of either M13, an unrelated filamentous phage, or G4, a ØX-type phage.     

Control of derepressed β-galactosidase synthesis inEscherichia coli

1. The synthesis of β-galactosidase in a constitutive mutant ofEscherichia coli (ML 308, i^-z⁺y⁺a⁺) responds to the nutritional environment. Repression can be reversed by cyclic AMP.
2. The greatest degree (%) of repression by metabolisable compounds is obtained when cells utilising glycerol (0%) are given, in addition, pyruvate (67%), serine (57%) which can be converted to pyruvate, or substrates of phosphotransferase systems (20–40%) which liberate pyruvate in their operation. Furthermore, pyruvate represses β-galactosidase synthesis in a phosphoenolpyruvate synthaseless mutant. Pyruvate, however, does not repress in a pyruvate dehydrogenaseless mutant and it follows that pyruvate itself is not the agent of repression.
3. Raffinose, a non-metabolisable galactoside, represses synthesis of β-galactosidase during growth on glycerol. Over a wide range, repression depends on raffinose concentration as does a lowered pool of ATP, rate of oxygen consumption and growth rate. All these parameters are inter-related but, in particular, β-galactosidase synthesis depends on the size of the ATP-pool presumably because this also limits synthesis of cyclic AMP under these conditions.

     

Comparative reactivities of diolepoxide metabolites of carcinogenic hydrocarbons with phiX174 viral DNA.

The ØX174 DNA assay system developed earlier is utilized to determine the comparative reactivities with nucleic acid of the diolepoxide metabolites of a series of polycyclic aromatic hydrocarbons varying in carcinogenic potency. The infectious ØX174 viral DNA is exposed to the hydrocarbon derivative for 10 min., then infectivity of the treated DNA is assayed by incubation with $\text{E.}\text{coli}$ spheroplasts, counting plaque formation on agar plates. The bay region diolepoxides of benzo[a]-pyrene, chrysene, and dibenz[a,h]anthracene, implicated as the ultimately active carcinogenic metabolites of the parent hydrocarbons, exhibit potent viral inhibitory activity. On the other hand no correlation is evident between viral inhibitory activity and either the location of the diolepoxide function in a bay region or the theoretically calculated β-delocalization energies (ΔE_deloc.) of the carbonium ion arising from opening the epoxide ring. The significance of these findings with respect to theories of carcinogenesis is discussed.     

The separation of phage promoter from bacterial lac promoter for beta-galactosidase expression in transducing phage lambda plac5.

The replication defective transducing phage λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 carries a portion of the $\text{E.}\text{coli}\text{lac}$ operon in the $\text{b}$2 region of the lambda phage. This $\text{lac}$ operon segment contains the $\text{lac}$ promoter, the $\text{lac}$ operator, and the β-galactosidase $\text{z}$ gene, but does not contain the $\text{lac}$ repressor $\text{i}$ gene. The $\text{z}$ gene can be expressed from both the inserted $\text{lac}$ promoter and the phage promoter. When $\text{E.}\text{coli}$ strain 594 ($\text{z}$^?, $\text{i}$⁺) or JC6256 (Δ$\text{lac}$) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ⁺) or JC6256 (λ⁺) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted $\text{lac}$ promoter.The ability to separate the phage promoter from the inserted $\text{lac}$ promoter for β-galactosidase expression will simplify the interpretation whenever λp$\text{lac}$5 is used.     

Culic 3', 5', -adenosine monophosphate and catabolic repression in Escherichia coli.

Several strains of $\text{E. coli}$ were grown on different sources of carbon and β-galactosidase activity as well as intracellular and extracellular concentrations of c-AMP were determined. There was a good (inverse) correlation between extracellular concentrations of c-AMP and the intensity of catabolite repression, whereas the relationship between intracellular c-AMP levels and catabolite repression was not clear-cut.     

Growth of phages lambda, phiX174, and Ml3 requires the dnaZ (previously dnaH) gene product of Escherichia coli

A functional $\text{dnaZ}$ (previously designated $\text{dnaH}$) product, known to be involved in DNA polymerization, is required for phage λ, ØX174, and M13, but not T4 or T7, growth.     

Structure of an intermediate in the replication of bacteriophage φX174 deoxyribonucleic acid: The initiation site for DNA replication

Replicative form DNA composed of a closed complementary strand and a discontinuous viral strand has been isolated from cells infected with bacteriophage φX174 during the period of single-strand DNA synthesis. This RFII DNA was degraded by the restriction enzyme from Hemophilus influenzae, endonuclease R, and the products analyzed by polyacrylamide gel electrophoresis. The results indicate that there are two types of discontinuity in the viral strands of these molecules: (1) 65% of the molecules contain a gap, which causes a discrete increase in mobility of a specific restriction enzyme fragment, R3. This gap can be selectively repaired with Escherichia coli DNA polymerase I and nucleoside triphosphates, but the molecules are not converted to RFI by addition of E. coli polynueleotide ligase to the reaction mixture. Approximately 30 moles of radioactive TTP are incorporated per mole of RF DNA. (2) 35% of the RF molecules contain a discontinuity, which does not result in a detectable change in mobility of any restriction enzyme fragment. These RF molecules can be converted to RFI by the action of ligase and polymerase I in the presence of nucleoside triphosphates, with incorporation of only approximately one mole of radioactive TTP, specifically into fragment R3, per mole of RF DNA.When the reaction of late RFII DNA and polymerase I is allowed to proceed beyond the repair of the discontinuity, radioactive nucleotides are incorporated into endonuclease R fragments adjacent to R3 in the 5′ → 3′ direction. This technique was utilized to determine a partial order of endonuclease R fragments in φX174.These results suggest that the synthesis of single-strand DNA is initiated from a unique point in cistron A and proceeds clockwise round the φX174 genetic map (cistron order: ABCDEFGH). A comparison of these results with other studies on φX174 suggests that DNA synthesis in all stages of φX174 replication may be initiated from a specific locus on the genome, at or near cistron A.     

DNA postreplication repair gap filling in temperature-sensitive dnaG, dnaC, dnaA mutants of Escherichiacoli

Gaps in daughter-strand deoxyribonucleic acid (DNA) synthesized after exposure of wild-type $\text{Escherichia}\text{coli}$ to ultraviolet light are filled during reincubation. In this study the $\text{dnaG}\text{,}\text{dnaC}$, and $\text{dnaA}$ gene products have been examined for their role in postreplication repair. These gene products are unique in their specific control of certain types of DNA synthesis: initiation of rounds of replication and chain propagation. Initiation of rounds of replication is not essential to gap filling; however, chain propagation by short DNA piece initiation appears to be essential for gap filling.     

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.     

Regulation of the in vitro synthesis of the alpha-peptide of beta-galactosidase directed by a restriction fragment of the lactose operon.

The regulation of the $\text{in vitro}$ synthesis of the N-terminal portion of the β-galactosidase molecule (α-peptide) has been investigated using DNA fragments of the lactose operon as template. DNA fragments of about 789 base pairs were isolated after endonuclease (Hin II) digestion of either λplac5, λh80dlacp^s or λh80dlacUV5 phage DNA or DNA from the recombinant plasmid PMC3. The regulation of the expression of these fragments is similar to that observed for the synthesis of β-galactosidase using total phage or plasmid DNA as template, indicating that the regulatory regions on the fragments are intact and functional. Thus, the synthesis of the α-peptide required an inducer due to the presence of $\text{lac}$ repressor in the $\text{E. coli}$ S-30 extract used. In addition a dependency on adenosine 3′,5′-cyclic monophosphate (cAMP)¹ for α-peptide synthesis was obtained with the fragments isolated from λplac5 and λh80dlacp^s DNAs, whereas little effect of cAMP was seen with the fragment isolated from λh80dlacUV5 phage DNA or PMC3 plasmid DNA containing a UV5 promotor region. However, a significant difference in the effect of guanosine-3′-diphosphate-5′-diphosphate (ppGpp) was observed. With the total phage DNA as template, ppGpp resulted in a 2–4 fold stimulation whereas with the fragment, or PMC3 plasmid DNA, directed synthesis of the α-peptide no significant stimulation by ppGpp was seen.     

Properties of iron-sulfur protein isolated from Pseudomonas ovalis.

Mutants of $\text{Escherichia coli}$ C were selected for resistance towards a set of cell wall LPS core specific bacteriophages, including øX174. Increasingly deficient LPS's from wt and mutant $\text{E. coli}$ C were tested for inactivation of øX174, and the core oligosaccharides were subjected to structural analysis by methylation/g.l.c./m.s. Loss of the terminal galactose in the following basic structure of the $\text{E. coli}$ C wt core was found to lead to adsorption resistance towards øX174:

     

An Rts1-derivative plasmid conferring UV sensitivity on Escherichia coli host

A deletion mutant was isolated from a kanamycin resistance R plasmid Rtsl. This mutant plasmid, pTW20, was found to enhance the lethal effect of UV irradiation on $\text{Escherichia}\text{coli}$ host, especially at 42°C. A cloning experiment with pTW20 DNA demonstrated that the gene, $\text{puv}$, being responsible for the UV sensitivity was located on the kanamycin resistance gene containing $\text{Bam}$H1 fragment of pTW20. This fragment conferred a sensitivity to methyl methane sulfonate on its host along with the sensitivity to UV, suggesting that a reapir process of the host chromosome is impaired by the presence of $\text{puv}$.     

Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by $\text{E. coli}$ DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I $\text{amber}$ mutants.     

Cloning of the replication origin from Drosophila virilis mitochondrial DNA.

Mitochondrial DNA from $\text{Drosophila}$ contains high “A+T”-rich region. Its DNA replication starts in the “A+T”-rich region and proceeds unidirectionally around the molecule. In order to determine precise location of the DNA replication origin and elucidate unique feature of its nucleotide sequence, the “A+T”-rich region of mitochondrial DNA from $\text{Drosophila}\text{virilis}$ has been cloned in $\text{Escherichia}\text{coli}$. The chimeric plasmid DNA containing the “A+T”-rich region stimulates $\text{in}\text{vitro}$ DNA replication system from $\text{Drosophila}\text{virilis}$ mitochondria about ten fold higher than the parental plasmid DNA, as does native mitochondrial DNA.     

Synthesis of the E. coli pyruvate dehydrogenase complex: non-dependence on 3'-5'-cyclic AMP

A determination of the level of the pyruvate dehydrogenase complex in a 3′–5′-c-AMP deficient mutant of $\text{E.}$$\text{coli}$ K12 has been carried out. The deficiency has no effect on specific activities for derivatives carrying either the inducible genes for two components of the complex or constitutive mutants. We conclude that synthesis of the complex is not sensitive to catabolite repression.     

Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins.

A novel iron uptake system was observed in pseudorevertants of $\text{Escherichia}$,$\text{coli}$ strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.