Phosphoryl Termini Research Articles

Sensitive detection of formamidopyrimidine-DNA glycosylase activity based on target-induced self-primed rolling circle amplification and magnetic nanoprobes.

We developed a novel approach to determine formamidopyrimidine DNA glycosylase (FPG) activity by taking advantage of target-induced self-primed rolling circle amplification (RCA) and magnetic nanoprobes. Herein, a unique nick (8-oxoguanine, 8-oxoG) was positioned in duplex DNA containing P-circle and P1, which together serve as a FPG substrate, RCA template, and RCA primer probe. The presence of FPG specifically binds 8-oxoG and cleaves the P-circle into two parts, producing 5'-phosphoryl termini. A phosphodiester bond between the 5'-phosphoryl and 3'-hydroxyl termini was formed with the addition of T4 DNA ligase, producing an unnicked circular strand. Using the unnicked strand as the RCA template, the P1 hybridized with the circle probe as a primer will trigger the RCA process. The RCA reaction produces amounts of long tandem-repeat DNA tiles with multiple recognizing regions for the FAM modified DNA probes (FP) and biotin-modified DNA probes (BP). With the streptavidin-biotin interaction, the BPs and FPs can be easily immobilized on the surface of streptavidin-modified magnetic microbeads (MBs). Due to the RCA enhanced and highly-concentrated fluorescence accumulation on the MBs, an ultralow detection limit of 1.033 U mL-1 for FPG was obtained. Combined with the high tolerance capability of human blood serum owing to magnetic isolation, the FPG assays in human blood serum were also obtained using fluorescence and confocal laser scanning microscopy. These results indicate that this robust self-primed RCA combined with magnetic nanoprobes is an excellent candidate for quantitatively monitoring the FPG activity responsible for DNA oxidative damage-related clinical diagnosis and therapy.

Facile and sensitive fluorescence sensing of alkaline phosphatase activity using NMM/G-quadruplex

Herein, we are presenting an approach that utilizes the λ exonuclease (λ exo) cleavage reaction in combination with the formation of G-quadruplex, thereby providing a label-free fluorometric tool for simply and accurately determining alkaline phosphatase (ALP) activity and inhibition. A hairpin probe (HP) with 5′-phosphoryl termini and 3′-end containing a G-rich region, is designed. Taking advantage of the efficient enzyme reactions, namely the λ exo cleavage reaction, the G-rich DNA fragment is released from HP and folds into a stable G-quadruplex in the presence of potassium ions, thus greatly enhance the fluorescence of N-methyl mesoporphyrin IX (NMM) (a specific G-quadruplex binder). However, in the presence of ALP, the 5′-phosphoryl of the HP is dephosphorylated. The yielding 5′-hydroxyl end product hampers the λ exo cleavage reaction. HP maintains its stem-loop structure. Thus the formation of the G-quadruplex is prohibited, and this results in weak fluorescence of NMM. The fluorescence intensity exhibits a linear correlation to ALP concentration in the range of 1–50 U/L with a detection limit of 0.75 U/L. Additionally, inhibition effect of sodium orthovanadate has also been investigated. This study offers a simple yet sensitive method for ALP activity assay.

Fluorescent assay for alkaline phosphatase activity based on graphene oxide integrating with λ exonuclease

A novel fluorescence turn-on strategy for the alkaline phosphatase (ALP) assay is developed based on the preferential binding of graphene oxide (GO) to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) coupled with λ exonuclease (λ exo) cleavage. Specifically, in the absence of ALP, the substrate-dsDNA constructed by one oligonucleotide with a fluorophore at the 3′-end (F-DNA) and its complementary sequence modified with a 5′-phosphoryl termini (p-DNA), is promptly cleaved by λ exo, and the resulting F-DNA is adsorbed on GO surface, allowing fluorescence quenching. Whereas the introduction of ALP leads to the hydrolysis of the P-DNA, and the yielding 5′-hydroxyl end product hampers the λ exo cleavage, inducing significant fluorescence enhancement due to the weak binding of dsDNA with GO. Under the optimized conditions, the approach exhibits high sensitivity and specificity to ALP with a detection limit of 0.19 U/L, and the determination of ALP in spiked human serum samples has also been realized. Notably, this new approach not only provides a novel and sensitive platform for the ALP activity detection but also promotes the exploitation of the GO-based biosensing for the detection of the protein with no specific binding element, and thus extending the GO-based sensing applications into a new field.

Sensitive electrochemical assay for T4 polynucleotide kinase activity based on dual-signaling amplification coupled with exonuclease reaction

A novel electrochemical strategy for simple and accurate monitoring of the activity and the inhibition of T4 polynucleotide kinase (PNK) is developed by coupling the λ exonuclease (λ exo) cleavage reaction with a dual-signaling amplification method. In the presence of PNK and adenosine triphosphate, the 5′-hydroxyl group of the substrate DNA is phosphorylated, initiating the hydrolysis of the 5′-phosphoryl termini catalyzed by λ exo, and by combining the resulting G–quadruplex–hemin signal-on probe and the ferrocene signal-off probe, a dual-signaling amplification strategy is realized. This approach exhibits a low detection limit of 0.02U/mL for PNK activity analysis, which is lower than or comparable to that of the previously reported PNK assays. Additionally, the inhibition effects of two known PNK inhibitors, namely ammonium sulfate and sodium hydrogen phosphate, have also been successfully detected. This new dual-signaling electrochemical biosensor provides a facile platform for the detection of the activity and inhibition of nucleotide kinases. With further exploration, it is possible to extend the as-proposed electrochemical biosensor to the detection of other analytes.

RNA Ligases

T4 RNA ligase 1 catalyzes the ATP-dependent covalent joining of single-stranded 5'-phosphoryl termini of DNA or RNA to single-stranded 3'-hydroxyl termini of DNA or RNA. T4 RNA ligase 2 also catalyzes the joining of a 3'-hydroxyl terminus of RNA to a 5'-phosphorylated RNA or DNA; unlike T4 RNA ligase 1, this enzyme prefers double-stranded substrates. A truncated form of T4 RNA ligase 2 requires a pre-adenylated substrate for ligation. This unit describes specific reaction conditions, as well as applications such as radioactive labeling of the 3' termini of RNA, circularizing oligodeoxyribonucleotides and oligoribonucleotides, ligating oligomers and nicks, creating hybrid and chimeric DNA/RNA molecules, and miRNA cloning.

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of – single strand preferential – deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M r of the purified enzyme determined by native PAGE was 67 000 and it is a tetramer and each protomer consists of two unidentical subunits of M r 21 000 and 13 000. It is an acidic protein with a p I of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg 2+, Mn 2+ and Co 2+ for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg 2+ was 40°C, it showed higher optimum temperature (45°C) for dsDNA hydrolysis in presence of Mn 2+ and Co 2+. Nuclease Rsn was inhibited by divalent cations like Zn 2+, Cu 2+ and Hg 2+, inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3′- or 5′-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3′-hydroxyl and 5′-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.

DNA binding properties and processive proofreading of herpes simplex virus type 1 DNA polymerase

The DNA binding properties of herpes simplex virus type 1 DNA polymerase (HSV pol), an alpha-like DNA polymerase, were investigated using an optimized band-shift assay. With linear double-stranded DNA (dsDNA), HSV pol formed two complexes. The favored DNA template was dsDNA with protruding 5′-phosphoryl termini. Stable binding of HSV pol was observed with a DNA hairpin containing a primer region of 9 bp of dsDNA, a 6-base loop and a 12-base 5′-terminal single-stranded extension. For the polymerization activity of HSV pol on poly(dT) an optimal primer length of 8 to 10 nucleotides was determined. The DNA binding event could be clearly separated from the enzymatic activities by its unique response to divalent cations and salt. Under ionic strength conditions where HSV pol exerts optimal polymerization activity in vitro, novel polymerase–DNA complexes were detected by band-shift analysis. These new complexes were similar while either in DNA polymerase or 3′,5′ exonuclease mode. Using a polymerase trap method and high-resolution polyacrylamide gel electrophoresis, HSV pol demonstrated internal switching from 3′,5′ exonuclease to polymerase-active mode during one DNA binding event. These results support the role of HSV pol as a true replicase, which proofreads without dissociating from the DNA template.

Transcription bypass or blockage at single-strand breaks on the DNA template strand: effect of different 3' and 5' flanking groups on the T7 RNA polymerase elongation complex.

We have studied the effects of single-strand breaks present on the template strand during T7 RNA polymerase transcription elongation. A synthetic DNA template with a T7 promoter was designed to contain a one-nucleotide gap at a defined location on the template strand. This gap, surprisingly, was efficiently bypassed by T7 RNA polymerase during transcription elongation, and the full-length transcript (FLT37) generated from the bypass event was shortened by one nucleotide compared to the full-length transcript (FLT38) generated from an intact, unbroken template strand. FLT37 did not contain any nucleotide insertions opposite to the gap, so that the RNA sequence downstream from the gap, although accurately transcribed, contained a single base deletion compared to FLT38. This, to our knowledge, is the first demonstration that the continuity of the DNA template strand is not a necessary requirement for DNA-dependent RNA polymerase transcription elongation. DNA templates with different 3' and 5' termini at the single-strand break site were also investigated in this study. One of these templates, 1/3P-4P, which contained 3'- and 5'-phosphoryl termini at the break site, efficiently blocked T7 RNA polymerase. A single phosphoryl group present on either the 3' or the 5' terminus of the break site did not efficiently block RNA polymerase progression, suggesting that the blockage observed with template 1/3P-4P is due to the repulsion between the two phosphoryl termini in the vicinity of the polymerase active site.

Mechanism of action of deoxyribonuclease II from human lymphoblasts

Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE-cellulose and heparin-agarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme. Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 microM Fe3+. Likewise we confirmed the presence of 3'-phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzed by Escherichia coli DNA polymerase I.

RNA Ligases

AbstractT4 RNA ligase, the product of the phage gene 63, is purified from phage‐infected cells. It catalyzes the ATP‐dependent covalent joining of single‐stranded 5'‐phosphoryl termini of DNA or RNA to single‐stranded 3'‐hydroxyl termini of DNA or RNA. This unit describes specific reaction conditions as well as applications such as radioactive labeling of 3' termini of RNA, circularizing deoxy‐ and ribo‐oligonucleotides, ligating oligomers for oligonucleotide synthesis, and stimulating the blunt‐end ligation activity of T4 DNA ligase.

DNA Base and Strand Damage in X-irradiated Monkey CV-1 Cells: Influence of Pretreatment Using Small Doses of Radiation

Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E. coli. The frequency and base sequence locations of the enzyme-sensitive sites were determined. Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines. The time for repair of half the single strand breaks was approximately 1.5 h. Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair. Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage. 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels. These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence. The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III.

Nuclease SP: a novel enzyme from spinach that incises damaged duplex DNA preferentially at sites of adenine

A novel endonuclease has been isolated from extracts of spinach leaves (Spinacia oleracea). The enzyme has been purified by a series of column chromatography steps and has a molecular size of approximately 43,000 daltons. The spinach endonuclease cleaved double stranded DNA damaged by ultraviolet light or cis-diamminedichloroplatinum (II) primarily at sites of adenine when end-labelled DNA fragments of defined sequence were employed as substrates. The nature of the structural distortion contained in damaged, duplex DNA appears to be an important determinant for endonuclease cleavage. DNA helical distortions produced by UV light-induced (6-4) pyrimidine-pyrimidone photoproducts, but not cyclobutane pyrimidine dimers are recognized by the enzyme. The DNA cleavage products generated by the enzyme contain 3'-hydroxyl and 5'-phosphoryl termini. Single stranded DNA and RNA are hydrolyzed by the spinach endonuclease. This enzyme, which we call nuclease SP, is similar in several respects to other single-strand-specific nucleases such as N. crassa and mung bean nucleases and may function in DNA repair and/or recombination events in spinach cells. Nuclease SP should be a useful tool for the analysis of (6-4) photoproducts occurring in duplex DNA.

Circular intermediates with missing nucleotides in the conversion of supercoiled or nicked circular to linear duplex DNA catalyzed by two species of BAL 31 nuclease

The extracellular nucleases from Alteromonas espejiana BAL 31 can catalyze the endonucleolytic and/or exonucleolytic hydrolysis of duplex DNA in response to a variety of alterations, either covalent or noncovalent, in DNA structure. The nuclease can exist as at least two kinetically and molecularly distinct protein species. The two species that have been studied, called the ‘fast’ ( F) and ‘slow’ ( S) nucleases, both readily convert negatively supercoiled DNAs to linear duplex molecules and accomplish this conversion through the formation of a circular duplex intermediate containing usually a single interruption in one strand. It is further shown that most of these intermediates contain gaps arising from the removal in a processive manner of one or more nucleotide residues after the introduction of the initial strand break (nick). Considering only the intermediates with gaps, the average number of missing residues is 6.3 ± 0.5 and 2.8 ± 0.3, respectively, for DNA acted upon by the F and S enzymes independently of the extent of conversion of supercoiled DNA. The nicks and gaps are bounded by 3′-hydroxyl and 5′-phosphoryl termini. When singly nicked circular DNA is used as the substrate, conversion to the linear duplex form occurs predominantly through a gapped circular intermediate with the same average numbers, within experimental error, of missing nucleotides for the respective nuclease species as found when supercoiled DNA is the substrate. The conversion to linear duplex DNA is much slower when nicked circular DNA is the substrate compared to that found when supercoiled DNA is the starting material.

DNA ligase from Drosophila melanogaster embryos. Substrate specificity and mechanism of action.

DNA ligase has been purified to homogeneity from 6-12 h Drosophila melanogaster embryos (Rabin, B. A., Hawley, R. S., and Chase, J. W. (1986) J. Biol. Chem. 261, 10637-10645). This enzyme had an apparent Km for ATP of 1.6 microM. Of a variety of nucleotides tested, only adenosine 5'-O-(3-thio)triphosphate could substitute for ATP in the joining reaction. The enzyme was competitively inhibited by dATP, with an apparent Ki of 2.3 microM. The apparent Km for DNA using p(dT)20 annealed with poly(dA) as substrate was 1.0 microM. Studies utilizing synthetic homopolymers showed that in addition to joining DNA to DNA, this enzyme could join the 5'-phosphoryl termini of RNA to the 3'-hydroxyl termini of DNA or RNA, when they were annealed with DNA. In addition, p(dT)7U could be joined when annealed with poly(dA). No joining was detected when RNA served as the template. Drosophila DNA ligase also catalyzed the joining of oligonucleotides containing a single mismatched nucleotide at their 3'-hydroxyl termini, as well as DNA containing short, complementary 5'-protruding ends, and in the presence of polyethylene glycol 6000, blunt-ended duplex DNA. The overall reaction mechanism was shown to be identical to that of the homologous prokaryotic DNA ligases. The joining reactions catalyzed by the Drosophila and T4 DNA ligases were shown to be reversible. Incubation of superhelical closed circular DNA molecules with the purified enzymes and AMP resulted in the production of a population of DNA molecules which had lost most, if not all, of their superhelical density.

Poly(deoxyadenylic-deoxythymidylic acid) damage by radiolytically activated neocarzinostatin.

The anaerobic reaction of poly(deoxyadenylic-deoxythymidylic acid) with neocarzinostatin activated by the carboxyl radical CO2-, an electron donor generated from gamma-ray radiolysis of nitrous oxide saturated formate buffer, has been characterized. DNA damage includes base release and strand breaks. Few strand breaks are formed prior to alkaline treatment; they bear 3'-phosphoryl termini. In contrast, most (66%) of the base release occurs spontaneously. DNA damage is highly (95%) specific for thymidine sites. Neither DNA-drug covalent adduct nor nucleoside 5'-aldehyde, which are major products in the DNA-nicking reaction initiated by mercaptans and oxygen, is formed in this reaction. Data are presented to show that the CO2(-)-activated neocarzinostatin intermediate is a short-lived free radical able to abstract hydrogen atoms from the C-1' and C-5' positions of deoxyribose. Attack occurs mostly (68%) at the C-1' position, producing a lesion whose properties are consistent with those of (oxidized) apyrimidinic sites.

Purification and Properties of an Acid Deoxyribonuclease from Rat Small Intestinal Mucosa

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.

Effect of ATP on the Friend Murine leukemia virus-associated endonuclease activity and the endonuclease activity of the avian myeloblastosis virus RNA-directed DNA polymerase.

The Mn2+-dependent endonuclease activity associated with the avian myeloblastosis virus RNA-directed DNA polymerase has been shown to be activated by ATP in the presence of Mg2+. In the presence of Mn2+ the endonucleolytic activity was stimulated about 3-fold by the addition of ATP. The earlier identified Mr = 40,000 Friend murine leukemia virus (F-MuLV)-associated endonuclease which functions in the presence of both Mg2+ and Mn2+ has also been shown to be similarly stimulated by ATP. For both endonuclease activities stimulation was only observed at ATP concentrations above 0.5 mM, and it did not increase upon elevating the ATP concentration above 2.5 mM. ADP and dATP also stimulated both activities, although not to the same extent as ATP. GTP had no apparent effect and AMP seemed to inhibit both activities. The effect ATP analogs had on the F-MuLV associated endonuclease activity could suggest that the endonuclease reaction in the presence of ATP might involve the cleavage of beta-gamma phosphate bonds in ATP. Neither adenyl-5'-yl imidodiphosphate nor (beta, gamma-methylene)adenosine 5'-triphosphate stimulated the activity, whereas significant stimulation was observed in the presence of (alpha, beta-methylene)adenosine 5'-triphosphate. Although no ATPase activity could be detected in the purified F-MuLV endonuclease preparation, the data do not exclude the possibility that ATP may be cleaved in amounts which are equivalent to the number of nicks introduced into DNA by the virus-associated endonuclease. In the presence of ATP and Mg2+ the F-MuLV-associated endonuclease nicked both supercoiled and linear DNA duplexes extensively, although the former was nicked more readily than the latter. Single-stranded DNA functioned poorly as a substrate. The nicks introduced by the enzyme contained a 5'-phosphoryl terminus and a 3'-hydroxyl group.

Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease. A three-step incision model.

The early steps of excision repair of cyclobutane pyrimidine dimers are investigated. It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus. This nick is not a substrate for T4 polynucleotide ligase. The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I. However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease. A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.

Binding of fluorescent analogs of GTP to the exchangeable nucleotide binding site of tubulin.

6 S tubulin from which the exchangeably bound guanine nucleotide had been removed was examined for its ability to bind two fluorescent analogs of GTP. One analog, (y-AmNS)GTP, contains the fluorophore l-aminonaphthalene 5-sulfonate attached to the y phosphate of GTP via a phosphoamidate bond. The second analog contains the same fluorophore attached to the sulfur atom of GTP-y-S via a 5-atom linker (GTP-y-SF). Both analogs bind to tubulin lacking nucleotide bound to the exchangeable site, as evidenced by significant increases in the anisotropy of nucleotide fluorescence in solutions containing tubulin. The Kd for the tubulinGTP-y-SF complex is 2.3 X M in buffers without glycerol. In solutions containing 25% glycerol both analogs bind about an order of magnitude more tightly (& for GTP-y-SF = 3.2 X M, Kd for (y-AmNS)GTP = 3.9 X M). Analysis of steady state and dynamic fluorescence anisotropies shows that the fluorophore of (y-AmNS)GTP bound to tubulin exhibits little rotational freedom and that the observed depolarization of fluorescence results largely from rotation of the tubulin-nucleotide complex. In contrast, GTP-y-SF bound to tubulin undergoes rapid rotational movement independent of the rotation of the tubulin-nucleotide complex. A number of GTP analogs were examined for their ability to displace tubulin-bound (y-AmNS)GTP, as measured by fluorescence anisotropy. These experiments show that some alterations of the phosphoryl side chain (GTP-y-S and guanosine-5’-(cu,P-methylene) triphosphate) have little effect on binding affinity, whereas others drastically weaken binding (guanosine5’-(P,y-methylene) triphosphate). The &y-methylene analog binds very weakly but is known to be able to promote assembly at concentrations sufficient to saturate the tubulin-exchangeable nucleotide-binding site. In contrast, the GTP analog containing a blocked phosphoryl terminus binds much more avidly than the plymethylene analog but fails to promote assembly. Thus, binding of a guanine nucleoside triphosphate is a necessary, but not sufficient, condition for promotion of tubulin assembly.

Evidence for the presence of chromatin-bound deoxyribonucleic acid phosphatase-exonuclease in Yoshida sarcoma ascites cells

An enzyme which cleaves the phosphoester bond of 3′-phosphoryl termini of DNA was isolated and purified from the chromatin of Yoshida sarcoma cells. The DNA phosphatase is specific for only 3′-phosphorylated DNA with a lesser activity for its single stranded form. Phosphoester bonds of various nucleotides, 3′-phosphorylated RNA and 5′-phosphorylated DNA were not hydrolysed by the enzyme. The DNA phosphatase required 10 mM MgCl 2, and was inactivated by 70 % with 1 mM ϱ-chloromercuribenzoate and completely by heat treatment at 70° for 5 min. Furthermore, an exonuclease activity could not be separated from the purified DNA phosphatase.