Reiji Okazaki

Mechanism of DNA chain growth: XVI. Analyses of RNA-linked DNA pieces in Escherichia coli with polynucleotide kinase

An improved method for the isolation of RNA-linked DNA pieces from Escherichia coli has been developed. The following results, obtained by end group labelling with T4 polynucleotide kinase, strongly suggest that the short RNA segment is covalently linked to the 5′ end of DNA. (1) After denaturation by dimethyl sulphoxide, all the labelled RNA bands at the position of DNA in a Cs 2 SO 4 equilibrium density gradient. (2) The number of 5′ termini of RNA equals the number of 5′-hydroxyl termini of DNA produced from the same preparation by alkaline hydrolysis. (3) After digestion with pancreatic DNAase, the 5′-terminally labelled RNA is resistant to periodate oxidation. This suggests that these molecules contain deoxyribonucleotides at their 3′ termini. The size of the majority of the RNA segments thus obtained ranges from mono- to trinucleotides. The cellular abundance of the RNA-linked DNA pieces has been estimated by selective polynucleotide kinase-catalysed labelling of the 5′-hydroxyl ends of DNA generated by alkaline hydrolysis. In the restrictive conditions RNA-linked DNA pieces accumulate in mutants defective in the 5′ → 3′ exonuclease and/or the polymerase activity of DNA polymerase I, but not in a DNA ligase mutant or in the wild-type control. This suggests that the removal of the RNA attached to the nascent DNA pieces requires the concerted action of both the 5′ → 3′ exonuclease and the polymerase activities of DNA polymerase I. The RNA-linked DNA pieces were hydrolysed with alkali and incubated with polynucleotide kinase and [γ- 32 P]ATP. When the DNA thus labelled is degraded to 5′-mononucleotides, the 32 P is found in all four deoxyribonucleotides.

Mechanism of DNA chain growth: XI. Structure of RNA-linked DNA fragments of Escherichia coli

Abstract The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ- 32 P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the 32 P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …

Mechanism of DNA chain growth. XV. RNA-linked nascent DNA pieces in Escherichia coli strains assayed with spleen exonuclease.

Abstract A new method for the detection and assay of RNA-linked nascent DNA pieces has been developed. The method relies on selective degradation by spleen exonuclease of radioactive 5′-OH terminated DNA produced from the pulse-labelled nascent pieces upon alkaline hydrolysis. Analysis with this method in wild type Escherichia coli has shown relatively high proportions of the RNA-linked molecules after shorter pulses and in the smaller pieces, supporting the transient nature of the RNA attachment to the nascent pieces. The RNA-linked nascent DNA pieces are accumulated by both E. coli pol Aex1 (defective in 5′ → 3′ exonuclease of DNA polymerase I) and E. coli pol A12 and pol A1 (defective in polymerase of DNA polymerase I), suggesting the requirement of the concerted action of both 5′ → 3′ exonuclease and polymerase of DNA polymerase I for the removal of the RNA attached to the nascent pieces. Most of the nascent DNA pieces accumulated by E. coli ligts 7 (defective in DNA ligase) are not linked to RNA, as expected from the direct role of DNA ligase in joining of the pieces. The analysis also has shown that a large portion of the nascent DNA pieces present in the cell under the normal steady-state conditions are not linked to RNA and that the level of the RNA-free DNA pieces is also increased in pol A mutants. These findings suggest that the removal of RNA from the nascent pieces is a relatively rapid process and the joining reaction is a rate-limiting step that requires the concurrent action of DNA polymerase and DNA ligase.

Mechanism of regulation of deoxythymidine kinase of Escherichia coli: I. Effect of regulatory deoxynucleotides on the state of aggregation of the enzyme☆

Abstract It is shown by preparative zone sedimentation in sucrose gradients and by Sephadex gel filtration that the feedback inhibitor (dTTP) and activator (dCDP, dCTP, dADP, etc.) of Escherichia coli deoxythymidine kinase may induce the “dimerization” of this enzyme. The molecular weights of the enzyme “monomer” and “dimer” are estimated by gel filtration to be approximately 42,000 and 89,000 to 91,000 respectively. Among various nucleotides, those which inhibit or activate the enzyme produce a marked increase of the sedimentation rate; little or no change in the sedimentation rate is detected with nucleotides having neither inhibitory nor activating effects. Furthermore, there is good correlation between the extent of inhibition by dTTP and the increase in the sedimentation rate produced by this nucleotide under a variety of conditions. Good correlation is also found between the extent of activation and the increase in the sedimentation rate found at various concentrations of dCDP (a strong activator) of dADP (a weak activator). These results are interpreted to indicate that “dimerization” is an essential change involved in both inhibitory and activating regulation of this enzyme by deoxynucleotides. The sedimentation coefficient of the enzyme increases to 5.3 to 5.5 s from 3.4 to 3.5 s on addition of an activator deoxynucleotide, whereas the sedimentation coefficient of 5.9 to 6.0 s is obtained when dTTP is added. This small difference in the extent of increase in the sedimentation rate may reflect differences in the conformation of the dimer formed in the presence of an activator and that formed in the presence of an inhibitor.

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase☆☆☆

Abstract The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-32P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, E. coli pol Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.

Mechanism of DNA chain growth: VII. Direction and rate of growth of T4 nascent short DNA chains

Abstract A method has been developed for isolating the 5′-phosphoryl terminal portion of a DNA chain as a large oligonucleotide by successive digestion of DNA with spleen DNase II and spleen phosphodiesterase followed by gel filtration. Using this method, it was shown that after a 6- to 30-second pulse with [3H]thymidine at 8 °C, the label incorporated into completed or nearly completed T4 nascent short DNA chains (about 9 s) of both strands is not in their 5′-phosphoryl termini. However, if cells are labelled for one minute or longer, the label is found in the 5′-terminal portion of these short chains. A similar pattern emerges at 14 °C. At this temperature, the [3H]thymidine label begins to be found in the 5′-terminal region about ten seconds after addition of [3H]thymidine. Thus, the T4 nascent short DNA chains of both strands are synthesized in the 5′ → 3′ direction and the time required for the formation of a single short chain is about one minute at 8 °C and ten seconds at 14 °C. These conclusions are confirmed by another method, in which the 5′-terminal portion of a DNA chain is degraded selectively to mononucleotides by treatment with alkaline phosphatase, pancreatic DNase I and spleen phosphodiesterase.

Mechanism of regulation of deoxythymidine kinase of Escherichia coli: II. Effect of temperature on the enzyme activity and kinetics☆

Abstract In the absence of a regulatory deoxynucleotide, the reaction of Escherichia coli deoxythymidine kinase is affected by temperature in an anomalous fashion. Under the ordinary experimental conditions, the maximum reaction rate occurs below 30 °C, and further increases in temperature result in a decrease in the rate. Such an effect is not seen in the presence of an activator or inhibitor deoxynucleotide (e.g. dCDP or dTTP), which appears to dimerize the enzyme. In the absence as well as in the presence of a regulatory deoxynucleotide, the reaction proceeds linearly at all temperatures between 0 and 43 °C. Therefore, the observed effect of temperature is not due to a progressive inactivation (denaturation) of the enzyme during the incubation. Evidence was also obtained that the temperature effect is reversible and instantaneous. The inhibitory influence of temperature is exaggerated by lowering the substrate concentrations and lessened by increasing them. This fact and the substrate saturation curves at various temperatures have clearly indicated that increasing temperature reduces the affinity of the enzyme for the substrates. It has also been noted that with decreasing temperature the sigmoidal nature of the saturation curve for ATP is weakened and approaches the normalized curve obtained in the presence of an activator. On the basis of these observations and those described in the preceding paper, it is postulated that E. coli deoxythymidine kinase is extremely temperature-sensitive in its “monomeric” form, and exists mostly in a less active state even at moderate temperatures. The enzyme is transformed into the temperature-insensitive “dimer” when a regulatory deoxynucleotide is added. This “dimer” molecule would have either an active or inactive conformation, depending on the specific nature of the regulatory deoxynucleotide with which it interacts.

Studies of deoxyribonucleic acid synthesis and cell growth in the deoxyriboside-requiring bacteria, Lactobacillus acidophilus: I. Biological and chemical nature of the intra-cellular acid-soluble deoxyribosidic compounds☆

Abstract Deoxyribosidic compounds in the acid-soluble fraction of the deoxyriboside-requiring bacteria, Lactobacillus acidophilus R-26, were studied using that bacteria as the test organism. It was shown that the majority of acid-soluble deoxyribosidic compounds in the cell were not nucleosides, and revealed a growth-supporting activity only after digestion with crude snake venom. The contents of these deoxyribosidic compounds were higher in the growing cells than in the resting cells. When cells were transferred to the medium with a limiting amount of deoxyriboside, a decrease in the level of the intracellular pool of these deoxyribosidic compounds was observed as DNA synthesis proceeded. These deoxyribosidic compounds revealed little growth effect before venom-treatment if tested alone, but if administered with sufficient thymidine they showed a growth effect about ten times as great as that observed in the absence of thymidine. These observations are taken to suggest that these deoxyribosidic compounds are intermediates in DNA synthesis. A preliminary attempt was made to isolate these compounds by using anion-exchange chromatography. Some properties of the deoxyribosidic compounds in a chromatographic fraction were studied. It is suggested that the main deoxyribosidic compound in the acid-soluble fraction of L. acidophilus is a deoxynucleotide-like compound which differs from the deoxymononucleotides hitherto found in the living material.

Mechanism of DNA chain growth. XIII. Evidence for discontinuous replication of both strands of P2 phage DNA.

By use of an ethanol/phenol mixture for stopping the pulse, joining of the labelled P2 short DNA chains during the subsequent operation is abolished more completely than with the ice and KCN mixture used previously (Kainuma-Kuroda & Okazaki, 1975). After stopping a brief [3H]thymidine pulse with this mixture, 60 to 65% of the radioactivity incorporated is recovered in the short chain fraction, while the rest is in DNA chains of one genome length or longer. Hybridization with the complementary phage DNA strands clearly indicates the presence of a small amount of nascent short chains of the L-strand. However, even after a very brief pulse, two-thirds of the pulse label incorporated into the L-strand is in DNA chains of one genome length or longer. If P2-infected cells of a polAts strain are pulse-labelled after transfer to a restrictive temperature, virtually all the label incorporated is found in short DNA chains. These short DNA chains, accumulated during inhibition of host DNA polymerase I, contain equal amounts of H and L-strand components. From these findings it is concluded that both strands of P2 DNA are replicated by the discontinuous mechanism but that the rate of joining of the short chains is very much faster in the L-strand than in the H-strand.

Studies of deoxyribonucleic acid synthesis and cell growth in the deoxyriboside-requiring bacteria, Lactobacillus acidophilus: III. Identification of thymidine diphosphate rhamnose

Abstract A new deoxynucleotide, thymidine diphosphate rhamnose, was isolated from Lactobacillus acidophilus R-26 which had been subjected to a treatment which induced an accumulation of deoxyribosidic compounds. The results presented indicate that in the nucleotide, thymidine-5′-monophosphate is linked to rhamnose-1-phosphate through a pyrophosphate bond. The possible metabolic function of the nucleotide is discussed.