φX174 DNA Research Articles

Inactivation and repair of double-stranded DNA damaged by the aziridinyl nitroimidazole RSU 1069

Incubation of RSU 1069 in the presence of biologically active double-stranded ΦX174 DNA resulted in, depending on pH, ionic strength and concentration of drug, inactivation of the DNA. A variety of lesions are induced including a high number of single-strand breaks and alkali-labile lesions, which are at most partly lethal. The main inactivating damage consists probably of base damage, induced by alkylation. A considerable part of the damage induced by RSU 1069 can be repaired by the various repair enzymes of the bacterial host of the ΦX174 DNA. Finally the damage (pattern) depends considerably on the ionic composition of the reaction solution, which can be explained by an equilibrium model presented in this paper.

Repair of damage induced by SR 4233

The benzotriazine di-N-oxide, SR 4233, was electrolytically reduced at constant potential at pH 4.0 at a reduction rate of 5%/hr under N 2 in the presence of φX174 DNA. During the reduction process, the biological infectivity of the bacterial phage was measured by a double transfection technique, either into the wild-type Escherichia coli strain, or into a series of seven mutants with specific, known defects in their capacity to repair DNA. The survival of φX174 was evaluated as an index of drug damage and from this we conclude that SR 4233 induces pH-dependent DNA damage in E coli, which is recognized and repaired primarily by the uvrC gene product and by the exonuclease III and endonuclease III gene products. These gene products act primarily upon and are responsible for the recognition of strand breaks and repair of oxidized and fragmented pyrimidine products, indicating that SR 4233 induces strand breaks in DNA resulting from oxidative damage to pyrimidines. As damage is maximized at acid pH, we further propose that the damage mechanism is a process of electron transfer from pyrimidine nucleotides in DNA (i.e., oxidation) to the protonated benzotriazine di-N-oxide one-electron radical anion.

Microelectrophoresis for the separation of DNA fragments.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo[a]pyrene diol epoxide.

The unwinding of supercoiled phi X174 RFI DNA induced by the tumorigenic (+) and non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) has been investigated by agarose slab-gel and ethidium titration tube gel electrophoresis. The differences in adduct conformations were verified by flow linear dichroism techniques. Both enantiomers cause a reversible unwinding by the formation of noncovalent intercalative complexes. The effects of covalently bound BPDE residues on the electrophoretic mobilities of the RF I DNA form in agarose gels were investigated in detail in the range of binding ratios rb approximately 0.0-0.06 (covalently bound BPDE residues/nucleotide). In this range of rb values, there is a striking difference in the mobilities of (+)-BPDE- and (-)-BPDE-adducted phi X174 DNA in agarose slab-gels, the covalently bound (+)-BPDE residues causing a significantly greater retardation than (-)-BPDE residues. Increasing the level of covalent adducts beyond rb approximately 0.06 in the case of the (+)-BPDE enantiomer, leads to further unwinding and a minimum in the mobilities (corresponding to comigration of the nicked form and the covalently closed relaxed modified form) at rb 0.10 +/- 0.01; at still higher rb values, rewinding of the modified DNA in the opposite sense is observed. From the minimum in the mobility, a mean unwinding angle (per BPDE residue) of theta = 12 +/- 1.5 degrees is determined, which is in good agreement the value of theta = 11 +/- 1.8 degrees obtained by the tube gel titration method. Using this latter method, values of theta = 6.8 +/- 1.7 degrees for (-)-BPDE-phi X174 adducts are observed. It is concluded that agarose slab gel techniques are not suitable for determining unwinding angles for (-)-BPDE-modified phi X174 DNA because the alterations in the tertiary structures for rb < 0.06 are too small to cause sufficiently large changes in the electrophoretic mobilities. The major trans (+)-BPDE-N2-guanosine covalent adduct is situated at external binding sites and the mechanisms of unwinding are therefore different from those relevant to noncovalent intercalative BPDE-DNA complexes or to classical intercalating drug molecules; a flexible hinge joint and a widening of the minor groove at the site of the lesion may account for the observed unwinding effects. The more heterogeneous (-)-BPDE-nucleoside adducts (involving cis and trans N2-guanosine, and adenosine adducts) are less effective in causing unwinding of supercoiled DNA for reasons which remain to be elucidated.

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213)

In this report, the types of DNA damage introduced by the ortho-quinone and the semi-quinone free radical of 4′- demethylepipodophyllotoxin-9-(4-6-O- ethylidene-β- d - glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) ΦX174 DNA have been examined in vitro. The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF ΦX174 DNA, respectively. The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation. The radical introduced, in addition, chemical DNA adducts. Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss ΦX174 DNA, while more than 40 were required for the inactivation of RF DNA. The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts [van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retèl J and Pinedo HM, Biochem Pharmacol 37: 3579–3589, 1988]. When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced. In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss ΦX174 DNA, resulted in a partial restoration of the biological activity. It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity.

Sequence-specific binding of a c-myc nuclear-matrix-associated region shows increased nuclear matrix retention after leukemic cell (HL-60) differentiation

HL-60 cells, a human promyelocytic leukemia cell line, contain amplified c-myc DNA sequences and mRNA transcripts. These cells can be induced to undergo macrophage differentiation by phorbol esters, which results in suppression of c-myc expression and cessation of cell proliferation. The nuclear matrix (NM), a nuclear skeleton resistant to DNase I digestion and high salt extraction, is proposed to be involved in DNA replication, gene regulation, and the correct distribution of DNA at mitosis. We have previously identified a nuclear-matrix-associated region (MAR) of the c-myc protooncogene to reside in a 1.4-kb region between Cla I and Eco RI restriction sites at the 3'-end of the gene. A 172-bp Dra I/Dra I subfragment of the 1.4-kb region was shown to be a major component of the MAR (myc-MAR), and this subfragment was demonstrated to be recognized by a nuclear protein (p25). In this report we demonstrate that phi X174 DNA, or the synthetic copolymers poly[d(G.C)] and poly[d(A.T)], are not effective suppressors of the binding of the myc-MAR to isolated NM, indicating that the binding sequence(s) are unique. We find that the addition of partially purified protein p25 increases the relative affinity of the myc-MAR for HL-60 NM in an in vitro assay system. NM isolated from HL-60 macrophages induced by phorbol esters retains significantly more myc-MAR DNA fragment in the presence of an excess amount of competitor DNA than does NM from untreated HL-60 cells. These data suggest that a change of the myc-MAR association with the NM occurs after monocytic differentiation of HL-60 cells.

Interaction of RSU 1069 and 1137 with DNA in vitro: Biological implications and mechanistic aspects

We have examined the capacity of the nitroimidazole aziridine antitumour drug RSU 1069 to react with DNA in vitro in order to get a better understanding of its mechanism of action. Moreover, we have utilized biologically active ΦX174 DNA to investigate the biological relevance of the chemical DNA modification induced by the drug. Incubation of RSU 1069 in the presence of single-stranded ΦX174 DNA resulted in extensive inactivation of the DNA, which is dependent on the concentration of drug and temperature. Only about 2% of the inactivating damage can be attributed to strand breakage. The main damage most probably consists of base damage, of which a part is non-lethal and alkali-labile which in turn can be converted into lethal lesion and subsequently into a break applying a post-incubation alkali treatment. Furthermore, from the dependence of the inactivation and also the formation of breaks on pH and ionic strength, it is concluded that the reaction most probably takes place between a protonated RSU 1069 and a negative DNA coil and that the damage pattern reflects the difference in reactivity of RSU 1069 with the phosphate groups and the bases in DNA. Comparison between RSU 1069 and its ring-open hydrolysis product RSU 1137 revealed that (lethal) damage induced in the DNA must be ascribed to the alkylating properties of the aziridine moiety.

Role of the semi-quinone free radical of the anti-tumour agent etoposide (VP-16-213) in the inactivation of single- and double-stranded ΦX174 DNA

The mechanism of action of the anti-tumour agent etoposide (VP-16-213) could involve its bioactivation to metabolites which can damage DNA. Active metabolites of etoposide, generated in vitro, are the 3',4'-dihydroxy-derivative (catechol) and its oxidation product, the ortho-quinone. The conversion of the catechol into the ortho-quinone (and vice versa) proceeds via formation of a semi-quinone free radical. We investigated the role of this radical species in the inactivation of biologically active single- (ss) and double-stranded (RF) phi X174 DNA. Since the formation of semi-quinone free radicals from the ortho-quinone of etoposide is pH dependent, experiments were performed, in which the ortho-quinone was incubated at pH 4, 7.4 and greater than or equal to 9. ESR measurements showed no formation of radical species from the ortho-quinone at pH 4, but an increased rate of generation of the primary semi-quinone free radical at pH values 7.4 to 10; at still higher pH values a secondary semi-quinone free radical was produced. HPLC analyses demonstrated chemical stability of the ortho-quinone at pH 4, but an accelerated decay was observed when the pH was elevated from 7.4 to 9, with its concomitant conversion into more polar components and into the catechol of etoposide. Ss phi X174 DNA, exposed to the ortho-quinone, was inactivated at an increasing rate at pH values increasing from 4 to 7.4 and subsequently to 9. RF phi X174 DNA was only significantly inactivated in incubations with the ortho-quinone at pH 4, not at pH values 7.4 and 9. From these data it is concluded that the primary semi-quinone free radical of etoposide may to a great extent be responsible for the ortho-quinone-induced ss phi X174 DNA inactivation, but that this radical species is not lethal towards RF phi X174 DNA.

Endo-exonuclease of Aspergillus nidulans

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.

Effects of Oxygen Radical Scavengers on the Inactivation of SS ΦX174 DNA by the Semi-Quinone Free Radical of the Antitumor Agent Etoposide

We have studied the effects of oxygen radical scavengers on the inactivation of ss phi X174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH greater than or equal to 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss phi X174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO, the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH greater than or equal to 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH. trapping but from trapping of O2-(.). DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH greater than or equal to 7.4. Potassium superoxide also stimulated phi X174 DNA inactivation by the ortho-quinone at pH less than or equal to 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the ortho-quinone of etoposide, thus indirectly influencing DNA inactivation.

Micrococcal nuclease digestion study of spermidine-condensed DNA

Spermidine-condensed lambda DNA tertiary structures have been studied by micrococcal nuclease digestion. Broad but discrete DNA bands were observed in gel electrophoresis experiments of digests at sizes of: 1003 ± 115 bp, 1972 ± 190 bp and 3100 ± 350 bp. These bands comprise an arithmetic series, similar to, but larger than, arithmetic DNA band series sizes we have observed previously in calf thymus and φx-174 DNA condensates. The 1003 bp monomer lambda DNA band size corresponds to wrapping B DNA once circumferentially about the toroidal-shaped tertiary structures, the predominant condensed structures present in these preparations, and is consistent with the measured electron microscopic dimensions for hydrated lambda DNA toruses previously presented. DNA fragment length stability was determined by release from the digested condensates. Fragments of 80–85 bp and sizes below are thermodynamically unstable in the lambda DNA condensates. This fragment size agrees well with a recent determination of the cooperativity size in DNA condensates.

Mutagenesis of φX174 am3 cs70 incorporated into the genome of mouse L-cells

The objective of our work with φX174 has been to develop a shuttle vector that can be used comparatively in bacterial cells, different types of mammalian cells, and possibly in the various tissues of transfenic mice, with a constant mechanism for detection and analysis of mutations independent of any host-cell type. Toward that end, we have efficiently rescued ΦX174 am3 cs70 that is host-silent and stably integrated into the genome of mouse L-cells. The particular mouse L-cell line contains tandem arrays, single copies, and fragments of ΦX that, upon restriction enzyme excision, can result in 5 potentially active copies per diploid genome. The excised ΦX DNA is recovered by column chromatography, ligated, and transfected into higly competent spheroplasts. The Rescue Efficiency, defined as the number of viable phages produced out of the total number of potentially recoverable copies, is approx. 10 −3. The Recovery Ratio, defined as the Rescue Efficiency for chromosomally-integrated phage DNA divided by the Rescue Efficiency for ΦX am3 cs70, is close to one. Mouse L-cels containing the integrated ΦX174 am3 cs70 were treated with 20 mM ethyl methanesulfonate. The reversion frequency of am3 among progeny phages rescued from treated cells was 1.4 × 10 −5 (193 revertants in 1.4 × 10 7 phages). This is significantly higher than the 5.8 × 10 −7 reversion frequency of am3 (7 revertants in 1.2 × 10 −7 phages) among progeny phages rescued from untreated cells.

SOS processing of unique oxidative DNA damages in Escherichia coli

φX174 replicative form (RF) I transfecting DNA containing thymine glycols (5,6-dihydroxy-5,6-dihydrothymine), urea glycosides or apurinic (AP) sites was used to study SOS processing of unique DNA damages in Escherichia coli. All three lesions can be found in DNA damaged by chemical oxidants or radiation and are representative of several common structural modifications of DNA bases. When φX DNA containing thymine glycols was transfected into host cells that were ultraviolet-irradiated to induce the SOS response, a substantial increase in survival was observed compared to transfection into uninduced hosts. Studies with mutants demonstrated that both the activated form of RecA and UmuDC proteins were required for this reactivation. In contrast, no increase in survival was observed when DNA containing urea glycosides or AP sites was transfected into ultraviolet-induced hosts. These data suggest that SOS-induced reactivation does not reflect a generalized repair system for all replication-blocking, lethal lesions but rather that the efficiency of reactivation is damage dependent. Further, we found that a significant fraction of potentially lethal thymine glycols could be ultraviolet-reactivated in an umuClex A rec A -independent manner, suggesting the existence of an as yet uncharacterized damage-inducible SOS-independent mode of thymine glycol repair.

Singlet molecular oxygen causes loss of biological activity in plasmid and bacteriophage DNA and induces single-strand breaks

Damage of plasmid and bacteriophage DNA inflicted by singlet molecular oxygen ( 1O 2) includes loss of the biological activity measured as transforming capacity in E. coli and single-strand break formation. Three different sources of 1O 2 were employed: (i) photosensitization with Rose bengal immobilized on a glass plate physically separated from the solution; (ii) thermal decomposition of the water-soluble endoperoxide 3,3′-(1,4-naphthylidene) dipropionate (NDPO 2); and (iii) microwave discharge. Loss of transforming activity was documented after exposing bacteriophage M13 DNA to 1O 2 generated by photosensitization employing immobilized Rose bengal, and with bacteriophage ΦX174 DNA, using the thermodissociable endoperoxide (NDPO 20) as a source of 1O 2. These findings are in agreement with experiments in which plasmid DNA pBR322 was exposed to a gas stream of 1O 2 generated by microwave discharge. The effects of 1O 2 quenchers and of 2H 2O indicate 1O 2 to be the species responsible. Strand-break formation in pBR322 and ΦX174, measured as an increase of the open circular form at the expense of the closed circular supercoiled form, was observed without alkaline treatment after exposing the DNA to 1O 2, using either agarose gel electrophoresis or sucrose gradient separation. The effect of quenchers and 2H 2O indicate the involvement of 1O 2 in DNA damage. We conclude that singlet oxygen can cause loss of biological activity and DNA strand breakage.

DNA polymerase α-DNA primase from human lymphoblasts

The DNA polymerase α-DNA primase complex from the human lymphoblast line HSC93 has been enriched to near homogeneity by using an immunoaffinity purification protocol which was developed earlier for the purification of the calf thymus enzyme (Nasheuer, H.-P. and Grosse, F. (1987) Biochemistry 26, 8458–8466). Immunoaffinity purified polymerase-primase from human cells consisted of four subunits displaying molecular weights of 195000 and 180000 for the DNA synthesizing α-subunit, of 68000 for the β-subunit, and of 55000 and 48000 for the primase-carrying γ- and δ-subunit, respectively. The isoelectric pH values for the individual subunits were estimated from non-equilibrium pH gradients to be between 5.9 and 5.7 for the α-subunit, at 5.5 for the β-subunit, and at 7.5 and 8.0 for the γ- and δ-subunit, respectively. The purified polymerase-primase converted single-stranded ΦX174 DNA into the double-stranded form in a primase-initiated reaction. During this process, 3–10 RNA primers were formed. RNA primers were about 11 nucleotides long. Elongation of existing RNA primers by the human polymerase-primase was semi-processive; following primer binding the DNA polymerase continuously incorporated 20 to 50 nucleotides, then it dissociated from the template DNA.

The orientation of DNA fragments in the agarose gels

A microscopic method of measuring the orientation of nucleic acids in the agarose gels is described. A nucleic acid undergoing electrophoresis is stained with the dye ethidium bromide and is viewed under high magnification with a polarization microscope. A high-numerical-aperture microscope objective is used to illuminate and to collect the fluorescence signal, and therefore the orientation of the minute quantities of nucleic acid can be measured: in a typical experiment we can detect the orientation of one-tenth of a picogram (10 −13 g) of DNA. Polarization properties of the fluorescent light emitted by the separate bands corresponding to different molecular weights of the DNA are examined. A linear dichroism equation relates the measured fluorescence to the mean orientation of the absorption dipole of the ethidium bromide (and therefore DNA) and to the extent to which it is disorganized. As an example, we measured the orientation of φX174 DNA RF HaeIII fragments undergoing electrophoresis in a field of 10 V/cm. Ethidium bromide bound to the fragments with an angle of the absorption dipole largely perpendicular to the direction of the electrophoretic current. The dichroism declined as the molecular weight of the fragments decreased which is interpreted as an increase in the degree of disorder for shorter DNA.

Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded ΦX174 DNA

We have studied the effects of the recently reported two new metabolites of the antitumor agent VP-16-213, the ortho-dihydroxy derivative or catechol and the ortho-quinone, on the biological activity of single-stranded and double-stranded ΦX174 DNA, the binding of the metabolites to calf thymus DNA and the conversion of the catechol into the ortho-quinone. Evidence was obtained for the oxidation of the catechol into the ortho-quinone and for the fact that the ortho-quinone is the metabolite of VP-16-213 responsible for its binding to rat liver microsomal proteins. The catechol and ortho-quinone of VP-16-213 were found to bind 7–9 times more strongly to calf thymus DNA than VP-16-213 itself. In contrast to the parent compound VP-16-213, the catechol as well as the ortho-quinone inactivated both single-stranded (ss) and double-stranded (RF) biologically active ΦX174 DNA. The mean T 37-values for inactivation of ss and RF ΦX174 DNA by 2.2 × 10 −4M catechol at 37° and pH 7.4 were 96 and 640 min, respectively. Reduction of the ortho-quinone by NADPH cytochrome P-450 reductase resulted in formation of the catechol. The system ortho-quinone/NADPH cytochrome P-450 reductase inactivated ss ΦX174 DNA with a mean T 37-value of 454 min, and this inactivation was inhibited by DMSO. The mean T 37-value for inactivation of ss ΦX174 DNA by 1.8 × 10 −4 M ortho-quinone at 37° and pH 4.0 was 24 min. The chemical stability of the ortho-quinone and the extent of inactivation of ss ΦX174 DNA by the ortho-quinone were both pH-dependent: at higher pH the ortho-quinone was less stable and gave less inactivation of DNA. The aqueous decomposition product(s) of the ortho-quinone formed at pH 7.4 inactivated ss ΦX174 DNA with a mean T 37-value of 175 min. The rate of inactivation of RF ΦX174 DNA by the ortho-quinone at pH 4.0 was twice as low as the rate of inactivation of ss ΦX174 DNA: T 37 = 49 min. When using excision repair deficient E.coli mutants (uvrA − or uvrC −), a higher inactivation of RF ΦX174 DNA was found: T 37 = 29 min for uvrA − E. coli, indicating that a part of the DNA damage introduced by the incubation with ortho-quinone is removed by excision repair. Neutral and alkaline sucrose gradient centrifugation of ss ΦX174 DNA incubated with the ortho-quinone at pH 4.0 revealed the absence of DNA-breakage, suggesting that introduction of strand breaks and alkali-labile sites does not play a role in the inactivation of DNA by the ortho-quinone. Adduct-formation probably plays a role in the inactivation of DNA by this metabolite.

ChemInform Abstract: DNA Strand Scission by Naturally Occurring 5‐Alkylresorcinols.

AbstractA dichloromethane extract of Hakea trifurcata is found to mediate the cleavage of ΦX174 DNA in the presence of Cu2+; bioassay‐guided fractionation affords the active constituents (I)‐(III), whose structures are determined by spectroscopy or independent syntheses.

Mechanism of replication of bacteriophage φX174 XXII. Site-specific mutagenesis of the [formula omitted] gene reveals that A ∗ protein is not essential for φX174 DNA replication

The A and A ∗ proteins of phage φX174 are encoded in the same reading frame in the viral genome; the smaller A ∗ protein is the result of a translational start signal within the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the φX174 A ∗ gene, previously cloned into pCQV2 under λ repressor control, into a TAG stop codon. The altered A ∗ gene was then inserted back into φX replicative form DNA to produce an amber mutant, φXamA ∗. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su + cells infected with φXamA ∗, φX gene A protein, altered at one amino acid, was synthesized at normal levels; A ∗ protein was not detectable. These observations indicate that the A ∗ protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of φX174 DNA or the packaging of the viral strand under the conditions tested.

Phosphodiesterase I in Human Urine: Purification and Characterization of the Enzyme

Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.